Therapeutic compositions comprising anti-NRP2 antibodies

ABSTRACT

Provided are affinity matured and humanized antibodies and antigen-binding fragments thereof that specifically bind to human neuropilin-2 (NRP2) polypeptides, including those that modulate binding interactions between human NRP2 and at least one NRP2 ligand, and which thereby modulate subsequent NRP2-mediated downstream signaling events, including related therapeutic compositions and methods for modulating NRP2 activity and treating diseases such as NRP2-associated diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 63/024,960, filed May 14, 2020; and U.S.Provisional Application No. 62/910,042, filed Oct. 3, 2019, each ofwhich is incorporated by reference in its entirety.

STATEMENT REGARDING THE SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ATYR_136_02US_ST25.txt. The text file is about261 KB, created on Oct. 2, 2020, and is being submitted electronicallyvia EFS-Web.

BACKGROUND Technical Field

Embodiments of the present disclosure relate to affinity matured andhumanized antibodies and antigen-binding fragments thereof thatspecifically bind to human neuropilin-2 (NRP2) polypeptides, includingthose that modulate binding interactions between human NRP2 and at leastone NRP2 ligand, and which thereby modulate subsequent NRP2-mediateddownstream signaling events, including related therapeutic compositionsand methods for modulating NRP2 activity and treating diseases such asNRP2-associated diseases.

Description of the Related Art

Recent research developments suggest that tRNA synthetases playimportant roles in cellular responses beyond their well-characterizedrole in protein synthesis. In particular, there is a growing recognitionthat tRNA synthetases participate in a range of previously-unrecognizedroles in responding to cellular stress and tissue homeostasis, in bothintracellular and extracellular environments.

Significant progress has been made in elucidating the role ofextracellular HARS derived proteins, including the identification of aputative cellular receptor, neuropilin-2 (NRP2 or NRP-2). Interactionsof HARS with NRP2 appear to be mediated by the N-terminal region ofHARS, and can lead to important changes in the cellular function ofNRP2.

Accordingly, the current discovery of this new pathway of regulationrepresents a previously unknown mechanism, which acts as a centralregulator of cellular processes, including, for example, axonalguidance, endocytosis, cell migration, proliferation, survival,apoptosis, lymphangiogenesis, cellular differentiation, and cellattachment with direct relevance to cancer initiation, growth,metastasis, and chemoresistance, as wells as muscular, vascular,neuronal, bone, and immune homeostasis. The deregulation of any of theseprocesses may lead to a spectrum of diseases, which may be addressed bythe development of anti-NRP2 antibodies that selectively target theneuropilin-2 axis. The present disclosure provides such antibodies andrelated embodiments.

BRIEF SUMMARY

Embodiments of the present disclosure include antibodies orantigen-binding fragments thereof that specifically bind to a humanneuropilin-2 (NRP2) polypeptide (anti-NRP2 antibody).

Certain embodiments include a therapeutic composition, comprising atleast one antibody or antigen-binding fragment thereof that specificallybinds to a human neuropilin-2 (NRP2) polypeptide (anti-NRP2 antibody),wherein the at least one antibody or antigen-binding fragment thereofcomprises: a heavy chain variable region (V_(H)) sequence that comprisescomplementary determining region V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3sequences selected from Table A1 or Table A3 and variants thereof whichspecifically bind to the human NRP2 polypeptide; and a light chainvariable region (V_(L)) sequence that comprises complementarydetermining region V_(L)CDR1, V_(L)CDR2, and V_(L)CDR3 sequencesselected from Table A1 or Table A3 and variants thereof whichspecifically bind to the human NRP2 polypeptide.

In some embodiments:

-   -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 1-3, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 4-6, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 7-9, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 10-12, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 13-15, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 16-18, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 19-21, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 22-24, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 25-27, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 28-30, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 31-33, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 34-36, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 34-39, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 40-42, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 57-59, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 60-62, respectively,        including variants thereof; or    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 63-65, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 66-68, respectively,        including variants thereof.

In some embodiments, the V_(H) sequence is at least 80, 85, 90, 95, 97,98, 99, or 100% identical to a sequence selected from Table A2,optionally wherein the V_(H) sequence has 1, 2, 3, 4, or 5 alterationsin the framework regions. In some embodiments, the V_(L) sequence is atleast 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequenceselected from Table A2, optionally wherein the V_(L) sequence has 1, 2,3, 4, or 5 alterations in the framework regions.

In some embodiments:

-   -   the V_(H) sequence comprises a sequence at least 80, 85, 90, 95,        97, 98, 99, or 100% identical to SEQ ID NO: 43, and the V_(L)        sequence comprises a sequence at least 80, 85, 90, 95, 97, 98,        99, or 100% identical to SEQ ID NO: 44;    -   the V_(H) sequence comprises a sequence at least 80, 85, 90, 95,        97, 98, 99, or 100% identical to SEQ ID NO: 45, and the V_(L)        sequence comprises a sequence at least 80, 85, 90, 95, 97, 98,        99, or 100% identical to SEQ ID NO: 46;    -   the V_(H) sequence comprises a sequence at least 80, 85, 90, 95,        97, 98, 99, or 100% identical to SEQ ID NO: 47, and the V_(L)        sequence comprises a sequence at least 80, 85, 90, 95, 97, 98,        99, or 100% identical to SEQ ID NO: 48;    -   the V_(H) sequence comprises a sequence at least 80, 85, 90, 95,        97, 98, 99, or 100% identical to SEQ ID NO: 49, and the V_(L)        sequence comprises a sequence at least 80, 85, 90, 95, 97, 98,        99, or 100% identical to SEQ ID NO: 50;    -   the V_(H) sequence comprises a sequence at least 80, 85, 90, 95,        97, 98, 99, or 100% identical to SEQ ID NO: 51, and the V_(L)        sequence comprises a sequence at least 80, 85, 90, 95, 97, 98,        99, or 100% identical to SEQ ID NO: 52;    -   the V_(H) sequence comprises a sequence at least 80, 85, 90, 95,        97, 98, 99, or 100% identical to SEQ ID NO: 53, and the V_(L)        sequence comprises a sequence at least 80, 85, 90, 95, 97, 98,        99, or 100% identical to SEQ ID NO: 54;    -   the V_(H) sequence comprises a sequence at least 80, 85, 90, 95,        97, 98, 99, or 100% identical to SEQ ID NO: 55, and the V_(L)        sequence comprises a sequence at least 80, 85, 90, 95, 97, 98,        99, or 100% identical to SEQ ID NO: 56;    -   the V_(H) sequence comprises a sequence at least 80, 85, 90, 95,        97, 98, 99, or 100% identical to SEQ ID NO: 69, and the V_(L)        sequence comprises a sequence at least 80, 85, 90, 95, 97, 98,        99, or 100% identical to SEQ ID NO: 70;    -   the V_(H) sequence comprises a sequence at least 80, 85, 90, 95,        97, 98, 99, or 100% identical to SEQ ID NO: 71, and the V_(L)        sequence comprises a sequence at least 80, 85, 90, 95, 97, 98,        99, or 100% identical to SEQ ID NO: 72; or    -   the V_(H) sequence comprises a sequence at least 80, 85, 90, 95,        97, 98, 99, or 100% identical to SEQ ID NO: 73, and the V_(L)        sequence comprises a sequence at least 80, 85, 90, 95, 97, 98,        99, or 100% identical to SEQ ID NO: 74.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to a full-length human NRP2polypeptide or a human NRP2 polypeptide selected from Table N1,optionally with an affinity of about 10 pM to about 500 pM or to about50 nM, or about, at least about, or no more than about 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 300, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM,or optionally with an affinity that ranges from about 10 pM to about 500pM, about 10 pM to about 400 pM, about 10 pM to about 300 pM, about 10pM to about 200 pM, about 10 pM to about 100 pM, about 10 pM to about 50pM, or about 20 pM to about 500 pM, about 20 pM to about 400 pM, about20 pM to about 300 pM, about 20 pM to about 200 pM, about 20 pM to about100 pM, about 20 pM to about 50 pM, or about 30 pM to about 500 pM,about 30 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM toabout 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM,or about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pMto about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90pM to about 900 pM, about 100 pM to about 1 nM, about 1 nM to about 5nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, or about 25 nM toabout 50 nM, optionally wherein the at least one antibody orantigen-binding fragment thereof specifically binds to the human NRP2polypeptide in its native form but does not substantially bind to thehuman NRP2 polypeptide in its denatured form.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof that specifically binds to at least one epitope in aneuropilin domain selected from one or more of the neuropilin b1 domain,neuropilin a1 domain, neuropilin a2 domain, neuropilin b2 domain,neuropilin c domain, neuropilin a1/a2 combined domain, neuropilin b1/b2combined domain, neuropilin a2/b1 combined domain, neuropilin b2/ccombined domain, neuropilin a2/b1/b2 combined domain, neuropilina2/b1/b2/c combined domain, neuropilin a1/a2/b1 combined domain,neuropilin a1/a2/b1/b2 combined domain, neuropilin a1/a2/b1/b2/ccombined domain, and the neuropilin b1/b2/c combined domain, optionallywith an affinity of about 10 pM to about 500 pM or to about 50 nM, orabout, at least about, or no more than about 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400,500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM, or optionallywith an affinity that ranges from about 10 pM to about 500 pM, about 10pM to about 400 pM, about 10 pM to about 300 pM, about 10 pM to about200 pM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, orabout 20 pM to about 500 pM, about 20 pM to about 400 pM, about 20 pM toabout 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM,about 20 pM to about 50 pM, or about 30 pM to about 500 pM, about 30 pMto about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or about 20pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about900 pM, about 100 pM to about 1 nM, about 1 nM to about 5 nM, about 5 nMto about 10 nM, about 10 nM to 25 nM, or about 25 nM to about 50 nM.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin a1 domain, the neuropilin a2 domain, and/or the neuropilina1a2 combined domain, including adjacent linker regions, optionally atabout residues;

-   -   (neuropilin a1 domain) 20-148, 30-141, 40-141, 50-141, 60-141,        70-141, 80-141, 90-141, 100-141, 110-141, 120-141, 130-141;        20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60,        20-50, 20-40, or 20-30 as defined by a human NRP2 precursor        sequence (see Table N1);    -   (neuropilin a2 domain) 142-280, 150-265, 160-265, 170-265,        180-265, 190-265, 200-265, 210-265, 220-265, 230-265, 240-265,        250-265, 260-265, 141-270, 141-260, 141-250, 141-240, 141-230,        141-220, 141-210, 141-200, 141-190, 141-180, 141-170, 141-160,        141-150, 200-250, 210-250, 220-250, 230-250, 200-240, 210-240,        220-240, 230-240, 227-247, 228-247, 229-247, 230-247, 231-247,        232-247, 233-247, 234-247, 235-247, 236-247; 227-246, 227-245,        227-244, 227-243, 227-242, 227-241, 227-240, 227-239, 227-238;        235-240, 236-239, 236-238, or residue 237 as defined by a human        NRP2 precursor sequence (see Table N1); or    -   (combined a1a2 domain) 20-280, 30-280, 40-280, 50-280, 60-280,        70-280, 80-280, 90-280, 100-280, 110-280, 120-280, 130-280,        140-280, 150-280, 160-280, 170-280, 180-280, 190-280, 200-280,        210-280, 220-280, 230-280, 240-280, 260-280, 270-280, 20-270,        20-260, 20-250, 20-240, 20-230, 20-220, 20-210, 20-200, 20-190,        20-180, 20-170, 20-160, 20-150, 20-140, 20-130, 20-120, 20-110,        20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30 as        defined by a human NRP2 precursor sequence (see Table N1).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin b1 domain, the neuropilin b2 domain, and/or the neuropilinb1/b2 combined domain, including adjacent linker regions, optionally atabout residues;

-   -   (neuropilin b1 domain) 299-420, 266-426, 280-426, 290-426,        300-426, 310-426, 320-426, 330-426, 340-426, 350-426, 360-426,        370-426, 380-426, 390-426, 400-426, 410-426, 420-426, 280-420,        280-410, 280-400, 280-390, 280-380, 280-370, 280-360, 280-350,        280-340, 280-330, 280-320, 280-310, 280-300, or 280-290 as        defined by a human NRP2 precursor sequence (see Table N1),        optionally wherein the epitope is a discontinuous epitope that        comprises one, two, or three of residues 299Y, 354N, and/or 416S        as defined by the human NRP2 precursor sequence;    -   (neuropilin b2 domain) 438-591, 450-591, 460-591, 470-591,        480-591, 490-591, 500-591, 510-591, 520-591, 530-591, 540-591,        550-591, 560-591, 570-591, 580-591, 438-590, 438-580, 438-570,        438-560, 438-550, 438-540, 438-530, 438-520, 438-510, 438-500,        438-490, 438-480, 438-470, 438-460, or 438-450 as defined by a        human NRP2 precursor sequence (see Table N1); or    -   (neuropilin b1/b2 combined domain) 266-591, 276-591, 286-591,        296-591, 306-591, 316-591, 326-591, 336-591, 346-591, 356-591,        366-591, 376-591, 386-591, 396-591, 406-591, 416-591, 426-591,        436-591, 446-591, 456-591, 466-591, 476-591, 486-591, 498-591,        508-591, 518-591, 528-591, 538-591, 548-591, 558-591, 568-591,        578-591, 588-591, 266-581, 266-571, 266-561, 266-551, 266-541,        266-531, 266-521, 266-511, 266-501, 266-491, 266-481, 266-471,        266-461, 266-451, 266-441, 266-431, 266-421, 266-411, 266-401,        266-391, 266-381, 266-371, 266-361, 266-351, 266-341, 266-331,        266-321, 266-311, 266-301, 266-291, 266-281, or 266-271 as        defined by a human NRP2 precursor sequence (see Table N1).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin a2/b1 combined domain and/or the neuropilin b2c combineddomain, including adjacent linker regions, optionally at about residues;

-   -   (neuropilin a2b1 combined domain) 149-437, 159-426, 169-426,        179-426, 189-426, 199-426, 209-426, 219-426, 229-426, 239-426,        249-426, 259-426, 269-426, 279-426, 289-426, 299-426, 309-426,        319-426, 329-426, 339-426, 349-426, 359-426, 369-426, 379-426,        389-426, 399-426, 409-426, 419-426, 149-436, 149-426, 149-416,        149-406, 149-396, 149-386, 149-376, 149-366, 149-356, 149-346,        149-336, 149-326, 149-316, 149-306, 149-296, 149-286, 149-276,        149-266, 149-256, 149-246, 149-236, 149-226, 149-216, 149-206,        149-196, 146-186, 146-176, 146-166, or 146-155 as defined by a        human NRP2 precursor sequence (see Table N1); or    -   (neuropilin b2c combined domain) 438-794, 448-794, 458-794,        468-794, 478-794, 487-794, 497-794, 507-794, 517-794, 527-794,        537-794, 547-794, 557-794, 567-794, 587-794, 597-794, 607-794,        617-794, 627-794, 637-794, 647-794, 657-794, 667-794, 677-794,        687-794, 697-794, 707-794, 717-794, 727-794, 737-794, 747-794,        757-794, 767-794, 777-794, 787-794, 427-794, 438-784, 438-774,        438-764, 438-754, 438-744, 438-734, 438-728, 438-714, 438-704,        438-694, 438-684, 438-674, 438-664, 438-654, 438-644, 438-634,        438-624, 438-614, 438-604, 438-596, 438-586, 438-576, 438-566,        438-556, 438-546, 438-536, 438-526, 438-516, 438-506, 438-494,        438-484, 438-474, 438-464, 438-454, 438-444 as defined by a        human NRP2 precursor sequence (see Table N1).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin c domain, including adjacent linker regions, optionally atabout residues 591-794, 600-794, 610-794, 620-794, 630-794, 640-794,650-794, 660-794, 670-794, 680-794, 690-794, 700-794, 710-794, 720-794,730-794, 740-794, 750-794, 760-794, 770-794, 780-794, 790-794, 591-790,591-780, 591-770, 591-760, 591-750, 591-740, 591-730, 591-720, 591-710,591-700, 591-690, 591-680, 591-670, 591-660, 591-650, 591-640, 591-630,591-620, 591-610, or 591-600 as defined by a human NRP2 precursorsequence (see Table N1).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin b1/b2/c combined domain, including adjacent linker regions,optionally at about residues 276-794, 286-794, 296-794, 306-794,316-794, 326-794, 336-794, 346-794, 356-794, 366-794, 376-794, 387-794,396-794, 406-794, 416-794, 426-794, 436-794, 446-794, 456-794, 466-794,476-794, 486-794, 496-794, 506-794, 516-794, 526-794, 536-794, 546-794,556-794, 566-794, 576-794, 586-794, 596-794, 606-794, 616-794, 626-794,636-794, 646-794, 656-794, 666-794, 676-794, 686-794, 696-794, 706-794,716-794, 726-794, 736-794, 746-794, 756-794, 766-794, 776-794, 786-794,266-794, 276-784, 276-774, 276-764, 276-754, 276-744, 276-734, 276-724,276-714, 276-704, 276-694, 276-684, 276-674, 276-664, 276-654, 276-644,276-634, 276-624, 276-614, 276-604, 276-594, 276-584, 276-574, 276-564,276-554, 276-544, 276-534, 276-524, 276-514, 276-504, 276-594, 276-584,276-574, 276-564, 276-554, 276-544, 276-534, 276-524, 276-514, 276-504,or 276-496 as defined by a human NRP2 precursor sequence (see Table N1).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in thejuxtamembrane domain (see Table N1), optionally selected from one ormore of the juxtamembrane domain of NRP2a (variant 1), the juxtamembranedomain of NRP2a (variant 2), the juxtamembrane domain of NRP2a (variant3), the juxtamembrane domain of NRP2b (variant 4), and the juxtamembranedomain of NRP2b (variant 5), including combinations thereof.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to a conformational epitope composedof two or more discontinuous epitope regions, optionally aconformational epitope comprising or consisting of:

-   -   (a) a first epitope region within the a1 domain, and second        epitope region within the a2 domain of the human NPR2        polypeptide;    -   (b) a first epitope region within the a1 domain, and second        epitope region within the b1 domain of the human NPR2        polypeptide;    -   (c) a first epitope region within the a1 domain, and second        epitope region within the b2 domain of the human NPR2        polypeptide;    -   (d) a first epitope region within the a1 domain, and second        epitope region within the c domain of the human NPR2        polypeptide;    -   (e) a first epitope region within the a1 domain, and second        epitope region within the juxtamembrane domain of the human NPR2        polypeptide selected from variant 1, 2, 3, 4 and 5;    -   (f) a first epitope region within the a2 domain, and second        epitope region within the b1 domain of the human NPR2        polypeptide;    -   (g) a first epitope region within the a2 domain, and second        epitope region within the b2 domain of the human NPR2        polypeptide;    -   (h) a first epitope region within the a2 domain, and second        epitope region within the c domain of the human NPR2        polypeptide;    -   (i) a first epitope region within the a2 domain, and second        epitope region within the juxtamembrane domain of the human NPR2        polypeptide selected from variant 1, 2, 3, 4 and 5;    -   (j) a first epitope region within the b1 domain, and second        epitope region within the b2 domain of the human NPR2        polypeptide;    -   (k) a first epitope region within the b1 domain, and second        epitope region within the c domain of the human NPR2        polypeptide;    -   (l) a first epitope region within the b1 domain, and second        epitope region within the juxtamembrane domain of the human NPR2        polypeptide selected from variant 1, 2, 3, 4 and 5;    -   (m) a first epitope region within the b2 domain, and second        epitope region within the c domain of the human NPR2        polypeptide;    -   (n) a first epitope region within the b2 domain, and second        epitope region within the juxtamembrane domain of the human NPR2        polypeptide selected from variant 1, 2, 3, 4 and 5; or    -   (o) a first epitope region within the c domain, and second        epitope region within the juxtamembrane domain of the human NPR2        polypeptide selected from variant 1, 2, 3, 4 and 5.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof modulates binding of the human NRP2 polypeptide to atleast one NRP2 ligand (optionally an NRP2 ligand selected from Table N2or Table N3 and/or a human histidyl-tRNA synthetase (HRS) polypeptideselected from Table H1, optionally a HRS splice variant selected fromone or more of SV9 (HRS(1-60)), SV11 (HRS(1-60)+(399-509)) and SV14(HRS(1-100)+(399-509)).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof is a blocking antibody which inhibits about or at leastabout 80-100% of the theoretical maximal binding between the human NRP2polypeptide and the least one NRP2 ligand, after pre-incubation with thehuman NRP2 polypeptide in a stoichiometrically equivalent amount,optionally about or at least about 80, 85, 90, 95, or 100% of thetheoretical maximal binding. In some embodiments, the at least oneantibody or antigen-binding fragment thereof is a partial blockingantibody which inhibits about or at least about 20-80% of thetheoretical maximal binding between the human NRP2 polypeptide and theat least one NRP2 ligand, after pre-incubation with the human NRP2polypeptide in a stoichiometrically equivalent amount, optionally aboutor at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80%of the theoretical maximal binding. In some embodiments, the at leastone antibody or antigen-binding fragment thereof specifically binds toan HRS polypeptide-interacting region of the NRP2 polypeptide, andmimics or agonizes one or more signaling activities of the HRSpolypeptide binding to the NRP2 polypeptide. In some embodiments, the atleast one antibody or antigen-binding fragment thereof specificallybinds to an HRS polypeptide-interacting region of the NRP2 polypeptide,and modulates binding/signaling activity between the NRP2 polypeptideand at least one NRP2 ligand. In some embodiments, the at least oneantibody or antigen-binding fragment thereof antagonizes thebinding/signaling activity between the NRP2 polypeptide and the at leastone NRP2 ligand.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof agonizes or enhances the binding/signaling activitybetween the NRP2 polypeptide and the at least one NRP2 ligand. In someembodiments, the at least one NRP2 ligand is selected from:

-   -   a VEGF selected from one or more of VEGF-A145, VEGF-A165,        VEGF-C, VEGF-D and PIGF-2;    -   a VEGF receptor (VEGFR) selected from VEGFR2 and VEGFR3;    -   a semaphorin selected from one or more of SEMA3-A, SEMA-3B,        SEMA-3C, SEMA-3D SEMA-3F, and SEMA-3G;    -   a plexin selected from one or more of plexin A1, A2, A3, A4, and        D1;    -   a growth factor selected from one or more of fibroblast growth        factor (FGF), hepatocyte growth factor (HGF), and platelet        derived growth factor (PDGF);    -   a growth factor receptor selected from one or more of a        fibroblast growth factor receptor (FGFR), a hepatocyte growth        factor receptor (HGFR), and a platelet derived growth factor        receptor (PDGF);    -   a galectin or a galectin receptor    -   a transcription factor selected from FAC1 and bromoprotein PHD        finger transcription factor;    -   an adaptor protein selected from one or more of GIPC1, GIPC2 and        GIPC3;    -   an integrin selected from Table N3, optionally one or more of        α_(v)β₁, α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈, α₆β₁ and α₆β₄;    -   a transforming growth factor beta selected from one or more of        TGFβ1, TGFβ2, TGFβ3, and their corresponding TGFβ receptors; and    -   an HRS polypeptide selected from Table H1, optionally an HRS        splice variant selected from one or more of HisRS^(N1),        HisRS^(N2), HisRS^(N3), HisRS^(N4) (SV9), HisRS^(N5),        HisRS^(C1), HisRS^(C2), HisRS^(C3), HisRS^(C4), HisRS^(C5),        HisRS^(C6), HisRS^(C7), HisRS^(C8) (SV11), and HisRS^(C9)        (SV14).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor and/or a semaphorin withoutsubstantially modulating the binding/signaling activity between the NRP2polypeptide and VEGFR2 or VEGFR3 or VEGF-C. In some embodiments, the atleast one antibody or antigen-binding fragment thereof antagonizes thebinding/signaling activity between the NRP2 polypeptide and a plexinreceptor and/or semaphorin without substantially modulating thebinding/signaling activity between the NRP2 polypeptide and a HRSpolypeptide. In some embodiments, the at least one antibody orantigen-binding fragment thereof antagonizes the binding/signalingactivity between the NRP2 polypeptide and a plexin receptor and/or asemaphorin without substantially modulating the binding/signalingactivity between the NRP2 polypeptide and a HRS polypeptide, and withoutsubstantially modulating the binding/signaling activity between the NRP2polypeptide and VEGFR2 or VEGFR3 or VEGF-C. In some embodiments, the atleast one antibody or antigen-binding fragment thereof antagonizes thebinding/signaling activity between the NRP2 polypeptide and VEGFR3without substantially modulating the binding/signaling activity betweenthe NRP2 polypeptide and a plexin receptor and/or a semaphorin. In someembodiments, the at least one antibody or antigen-binding fragmentthereof antagonizes the binding/signaling activity between the NRP2polypeptide and VEGFR3 or VEGF-C without substantially modulating thebinding/signaling activity between the NRP2 polypeptide and a differentligand, optionally an HRS polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor without substantially modulatingthe ligand binding of semaphorin 3 to NRP2. In some embodiments, theplexin receptor is selected from plexin A1, A2, A3, A4, and D1. In someembodiments, the semaphorin is selected from semaphorin 3B, 3C, 3D, 3F,and 3G.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope of at least 5contiguous amino acids within the human NRP2 a2 domain, wherein the atleast one antibody or antigen-binding fragment thereof selectivelyinhibits receptor dimerization between NRP2 and plexin A1 withoutsubstantially inhibiting dimerization between NRP2 and FLT4 (VEGFR3). Insome embodiments, the at least one antibody or antigen-binding fragmentthereof specifically binds to an epitope within amino acids 232-242 of ahuman NRP2 precursor (see Table N1). In some embodiments, the at leastone antibody or antigen-binding fragment thereof specifically binds to adiscontinuous epitope comprised within amino acids 299-416 the humanNRP2 b1 domain, wherein the at least one antibody or antigen-bindingfragment thereof selectively inhibits receptor dimerization between NRP2and FLT4 (VEGFR3) and KDR (VEGFR2) without substantially inhibitingdimerization between NRP2 and plexin A1.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope of at least 5contiguous amino acids within the human NRP2 b2 domain, wherein the atleast one antibody or antigen-binding fragment thereof inhibits receptordimerization between NRP2 and FLT4 (VEGFR3) and inhibits dimerizationbetween NRP2 and plexin A1. In some embodiments, the at least oneantibody or antigen-binding fragment thereof specifically binds to anepitope of at least 5 contiguous amino acids within the human NRP2 cdomain, wherein the at least one antibody or antigen-binding fragmentthereof inhibits receptor dimerization between NRP2 and plexin A1 andpartially inhibits dimerization between NRP2 and FLT4 (VEGFR3).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof has an affinity (Kd or EC₅₀) for each of (i) a humanNRP2 polypeptide and (ii) the corresponding region of a cynomolgusmonkey NRP2 polypeptide, wherein the affinity for (i) and (ii) is withinthe range of about 20 pM to about 200 pM, about 30 pM to about 300 pM,about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM toabout 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM,about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 0.4 toabout 1.2 nM, about 0.9 to about 5.5 nM, about 0.9 to about 5 nM, orabout 1 nM to about 10 nM. In some embodiments, the at least oneantibody or antigen-binding fragment thereof has an affinity (Kd orEC₅₀) for each of (i) a human NRP2 polypeptide and (ii) thecorresponding region of a murine NRP2 polypeptide, wherein the affinityfor (i) and (ii) is within the range of about 20 pM to about 200 pM,about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM toabout 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM,about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pMto about 1 nM, or about 1 nM to about 10 nM.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof binds selectively to the NRP2a isoform (optionallyvariants 1, 2, and/or 3 of Table N1) of NRP2, and does not substantiallybind to the NRP2b isoform (optionally variants 4 and/or 5 of Table N1).In some embodiments, the at least one antibody or antigen-bindingfragment thereof binds selectively to the NRP2b isoform (optionallyvariants 4 and/or 5 of Table N1), and does not substantially bind to theNRP2a isoform (optionally variants 1, 2, and/or 3 of Table N1). In someembodiments, the at least one antibody or antigen-binding fragmentthereof reduces the homo- or hetero-dimerization between NRP2polypeptides, optionally by about or at least about 20-100% (e.g., about20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, or 100%) afterpre-incubation of the anti-NRP2 antibody with the NRP2 polypeptides in asubstantially stoichiometrically equivalent amount, optionally in thepresence of an NRP2 ligand.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof enhances the homo- or hetero-dimerization between NRP2polypeptides, optionally by about or at least about 20-100% (e.g., about20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, or 100%) afterpre-incubation of the anti-NRP2 antibody with the NRP2 polypeptides in asubstantially stoichiometrically equivalent amount, optionally in thepresence of an NRP2 ligand. In some embodiments, the at least oneantibody or antigen-binding fragment thereof binds selectively to ahuman NRP2 polypeptide (see Table N1) relative to a murine NRP2polypeptide, optionally where its affinity for the human NRP2polypeptide is significantly stronger than its affinity for the murineNRP2 polypeptide, optionally by about or at least about 2, 5, 10, 20,30, 40, 50, 100, 500, or 1000-fold or more. In some embodiments, the atleast one antibody or antigen-binding fragment thereof binds to thehuman NRP2 polypeptide and does not substantially bind to the murineNRP2 polypeptide, optionally wherein the murine NRP2 polypeptide is aMus musculus NRP2 polypeptide. In some embodiments, the at least oneantibody or antigen-binding fragment thereof binds to an epitope in theb1 domain that comprises residues 299Y, 354N, and 416S, as defined by ahuman NRP2 precursor sequence (see Table N1).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof comprises an IgA (including subclasses IgA1 and IgA2),IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), or IgMFc domain, optionally a human Fc domain, or a hybrid and/or variantthereof. In some embodiments, the at least one antibody orantigen-binding fragment thereof comprises an IgG Fc domain with higheffector function in humans, optionally an IgG1 or IgG3 Fc domain. Insome embodiments, the at least one antibody or antigen-binding fragmentthereof comprises an IgG Fc domain with low effector function in humans,optionally an IgG2 or IgG4 Fc domain. In some embodiments, the at leastone antibody or antigen-binding fragment thereof comprises an IgG1 orIgG4 Fc domain, optionally selected from Table F1. In some embodiments,the at least one antibody or antigen-binding fragment thereof comprisesa modified IgG1 or IgG4 Fc domain which has altered binding to FcRn,optionally wherein the modified IgG1 or IgG4 Fc domain comprises any oneor more of YD (M252Y/T256D), DQ (T256D/T307Q), DW (T256D/T307W), YTE(M252Y/S254T/T256E), AAA (T307A/E380A/N434A), LS (M428L/N434S), M252Y,T256D/E, K288D/N, T307Q/W, E380C, N434FY, and/or Y436H/N/W mutations (EUnumbering), including combinations thereof.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof is a monoclonal antibody. In some embodiments, the atleast one antibody or antigen-binding fragment thereof is a humanizedantibody. In some embodiments, the at least one antibody orantigen-binding fragment thereof is an Fv fragment, a single chain Fv(scFv) polypeptide, an adnectin, an anticalin, an aptamer, an avimer, acamelid antibody, a designed ankyrin repeat protein (DARPin), aminibody, a nanobody, or a unibody.

In some embodiments, the therapeutic composition has a purity of atleast about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis withrespect to the at least one antibody or antigen-binding fragment, and issubstantially aggregate-free. In some embodiments, the therapeuticcomposition is substantially endotoxin-free. In some embodiments, thetherapeutic composition is a sterile, injectable solution, optionallysuitable for intravenous, intramuscular, subcutaneous, orintraperitoneal administration.

In some embodiments, the therapeutic composition further comprises atleast one additional agent selected from one or more of a cancerimmunotherapy agent, a chemotherapeutic agent, a hormonal therapeuticagent, and a kinase inhibitor. In some embodiments, the cancerimmunotherapy agent is selected from one or more of an immune checkpointmodulatory agent, a cancer vaccine, an oncolytic virus, a cytokine, anda cell-based immunotherapies. In some embodiments, the immune checkpointmodulatory agent is a polypeptide, optionally an antibody orantigen-binding fragment thereof or a ligand, or a small molecule. Insome embodiments, the immune checkpoint modulatory agent comprises

-   -   (a) an antagonist of a inhibitory immune checkpoint molecule; or    -   (b) an agonist of a stimulatory immune checkpoint molecule,    -   optionally wherein the immune checkpoint modulatory agent        specifically binds to the immune checkpoint molecule.

In some embodiments, the inhibitory immune checkpoint molecule isselected from one or more of Programmed Death-Ligand 1 (PD-L1),Programmed Death 1 (PD-1), Programmed Death-Ligand 2 (PD-L2), CytotoxicT-Lymphocyte-Associated protein 4 (CTLA-4), Indoleamine 2,3-dioxygenase(IDO), tryptophan 2,3-dioxygenase (TDO), T-cell Immunoglobulin domainand Mucin domain 3 (TIM-3), Lymphocyte Activation Gene-3 (LAG-3),V-domain Ig suppressor of T cell activation (VISTA), B and T LymphocyteAttenuator (BTLA), CD160, Herpes Virus Entry Mediator (HVEM), and T-cellimmunoreceptor with Ig and ITIM domains (TIGIT).

In some embodiments:

-   -   the antagonist is a PD-L1 and/or PD-L2 antagonist optionally        selected from one or more of an antibody or antigen-binding        fragment or small molecule that specifically binds thereto,        atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab        (MEDI4736);    -   the antagonist is a PD-1 antagonist optionally selected from one        or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto, nivolumab,        pembrolizumab, MK-3475, AMP-224, AMP-514, PDR001, and        pidilizumab;    -   the antagonist is a CTLA-4 antagonist optionally selected from        one or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto, ipilimumab, and        tremelimumab;    -   the antagonist is an IDO antagonist optionally selected from one        or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto, indoximod (NLG-8189),        1-methyl-tryptophan (1MT), β-Carboline (norharmane;        9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat;    -   the antagonist is a TDO antagonist optionally selected from one        or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto, 680C91, and LM10;    -   the antagonist is a TIM-3 antagonist optionally selected from        one or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto;    -   the antagonist is a LAG-3 antagonist optionally selected from        one or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto, and BMS-986016;    -   the antagonist is a VISTA antagonist optionally selected from        one or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto;    -   the antagonist is a BTLA, CD160, and/or HVEM antagonist        optionally selected from one or more of an antibody or        antigen-binding fragment or small molecule that specifically        binds thereto; and/or    -   the antagonist is a TIGIT antagonist optionally selected from        one or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto.

In some embodiments, the stimulatory immune checkpoint molecule isselected from one or more of OX40, CD40, Glucocorticoid-Induced TNFRFamily Related Gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and HerpesVirus Entry Mediator (HVEM).

In some embodiments:

-   -   the agonist is an OX40 agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto, OX86,        Fc-OX40L, and GSK3174998;    -   the agonist is a CD40 agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto, CP-870,893,        dacetuzumab, Chi Lob 7/4, ADC-1013, and rhCD40L;    -   the agonist is a GITR agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto, INCAGN01876,        DTA-1, and MED11873;    -   the agonist is a CD137 agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto, utomilumab,        and 4-1BB ligand;    -   the agonist is a CD27 agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto, varlilumab,        and CDX-1127 (1F5);    -   the agonist is a CD28 agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto, and TAB08;        and/or    -   the agonist is an HVEM agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto.

In some embodiments, the cancer vaccine is selected from one or more ofOncophage, a human papillomavirus HPV vaccine optionally Gardasil orCervarix, a hepatitis B vaccine optionally Engerix-B, Recombivax HB, orTwinrix, and sipuleucel-T (Provenge), or comprises a cancer antigenselected from one or more of human Her2/neu, Her1/EGF receptor (EGFR),Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE Receptor),MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growthfactor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40,CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR,CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1(IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA),guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrinα5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblastactivation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (orCA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA),NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamilymember 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), and mesothelin.

In some embodiments, the oncolytic virus selected from one or more oftalimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™),Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010),Senecavirus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102(Ad5/3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

In some embodiments, the cytokine selected from one or more ofinterferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and Granulocyte-macrophagecolony-stimulating factor (GM-CSF).

In some embodiments, the cell-based immunotherapy agent comprises cancerantigen-specific T-cells, optionally ex vivo-derived T-cells. In someembodiments, the cancer antigen-specific T-cells are selected from oneor more of chimeric antigen receptor (CAR)-modified T-cells, and T-cellReceptor (TCR)-modified T-cells, tumor infiltrating lymphocytes (TILs),and peptide-induced T-cells.

In some embodiments, the at least one chemotherapeutic agent is selectedfrom one or more of an alkylating agent, an anti-metabolite, a cytotoxicantibiotic, a topoisomerase inhibitor (type 1 or type II), and ananti-microtubule agent.

In some embodiments:

-   -   the alkylating agent is selected from one or more of nitrogen        mustards (optionally mechlorethamine, cyclophosphamide, mustine,        melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas        (optionally N-Nitroso-N-methylurea (MNU), carmustine (BCNU),        lomustine (CCNU), semustine (MeCCNU), fotemustine, and        streptozotocin), tetrazines (optionally dacarbazine,        mitozolomide, and temozolomide), aziridines (optionally        thiotepa, mytomycin, and diaziquone (AZQ)), cisplatins and        derivatives thereof (optionally carboplatin and oxaliplatin),        and non-classical alkylating agents (optionally procarbazine and        hexamethylmelamine);    -   the anti-metabolite is selected from one or more of anti-folates        (optionally methotrexate and pemetrexed), fluoropyrimidines        (optionally 5-fluorouracil and capecitabine), deoxynucleoside        analogues (optionally ancitabine, enocitabine, cytarabine,        gemcitabine, decitabine, azacitidine, fludarabine, nelarabine,        cladribine, clofarabine, fludarabine, and pentostatin), and        thiopurines (optionally thioguanine and mercaptopurine);    -   the cytotoxic antibiotic is selected from one or more of        anthracyclines (optionally doxorubicin, daunorubicin,        epirubicin, idarubicin, pirarubicin, aclarubicin, and        mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and        actinomycin;    -   the topoisomerase inhibitor is selected from one or more of        camptothecin, irinotecan, topotecan, etoposide, doxorubicin,        mitoxantrone, teniposide, novobiocin, merbarone, and        aclarubicin; and/or    -   the anti-microtubule agent is selected from one or more of        taxanes (optionally paclitaxel and docetaxel) and vinca        alkaloids (optionally vinblastine, vincristine, vindesine,        vinorelbine).

In some embodiments, the at least one hormonal therapeutic agent is ahormonal agonist or a hormonal antagonist. In some embodiments, thehormonal agonist is selected from one or more of a progestogen(progestin), a corticosteroid (optionally prednisolone,methylprednisolone, or dexamethasone), insulin like growth factors, VEGFderived angiogenic and lymphangiogenic factors (optionally VEGF-A,VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor(FGF), galectin, hepatocyte growth factor (HGF), platelet derived growthfactor (PDGF), transforming growth factor (TGF)-beta, an androgen, anestrogen, and a somatostatin analog. In some embodiments, the hormonalantagonist is selected from one or more of a hormone synthesisinhibitor, optionally an aromatase inhibitor or a gonadotropin-releasinghormone (GnRH) or an analog thereof, and a hormone receptor antagonist,optionally a selective estrogen receptor modulator (SERM) or ananti-androgen, or an antibody directed against a hormonal receptor,optionally cixutumumab, dalotuzumab, figitumumab, ganitumab,istiratumab, robatumumab, alacizumab pegol, bevacizumab, icrucumab,ramucirumab, fresolimumab, metelimumab, naxitamab, cetuximab,depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine,matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab,tomuzotuximab, zalutumumab, aprutumab ixadotin, bemarituzumab,olaratumab, or tovetumab.

In some embodiments, the kinase inhibitor is selected from one or moreof adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib,cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib,entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib,imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab,pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib,sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, andvemuafenib.

Also included are methods of treating a disease or condition in asubject in need thereof, comprising administering to the subject atherapeutic composition comprising at least one antibody orantigen-binding fragment thereof that specifically binds to a humanneuropilin-2 (NRP2) polypeptide, wherein the at least one antibody orantigen-binding fragment thereof modulates (e.g., interferes with)binding of the human NRP2 polypeptide to a human histidyl-tRNAsynthetase (HRS) polypeptide, optionally as a therapeutic compositiondescribed herein.

In some embodiments, the disease or condition is an NRP2-associateddisease or condition. In some embodiments, the disease or condition isselected from one or more of cancer and diseases and pathways associatedwith cancer, including cancer cell growth, initiation, migration,adhesion, invasion, chemoresistance, and/or metastasis; diseasesassociated with inflammation, autoimmunity, and related inflammatorydiseases, including diseases associated with inappropriate immune cellactivation or migration such as Graft versus host disease (GVHD);diseases associated with lymphatic development, lymphangioma,lymphangiogenesis, and lymphatic damage, including, for example, edema,lymphedema, secondary lymphedema, inappropriate fat absorption anddeposition, excess fat deposition, and vascular permeability; diseasesassociated with infections, including latent infections; diseasesassociated with allergic disorders/diseases, allergic responses,including, for example, chronic obstructive pulmonary disorder (COPD),neutrophilic asthma, antineutrophil cytoplasmic antibody(ANCA)-associated systemic vasculitis, systemic lupus erythematosus,rheumatoid arthritis, inflammasome-related diseases, and skin-relatedneutrophil-mediated diseases such as pyoderma gangrenosum; diseasesassociated with granulomatous inflammatory diseases, includingsarcoidosis and granulomas; diseases associated with fibrosis includingfibrotic diseases, fibrosis, endothelial to mesenchymal transition(EMT), and wound healing; diseases associated with inappropriate smoothmuscle contractility, smooth muscle compensation and decompensation, andinappropriate vascular smooth muscle cell migration and adhesion;diseases associated with inappropriate autophagy, phagocytosis, andefferocytosis; diseases associated with inappropriate migratory cellmovement; diseases associated with neuronal diseases, peripheral nervoussystem remodeling, and pain perception; and diseases associated withbone development and bone remodeling.

In some embodiments, the disease is a cancer, optionally wherein thecancer expresses or overexpresses NRP2, optionally wherein the cancerdisplays NRP2-dependent growth, NRP2-dependent adhesion, NRP2-dependentmigration, and/or NRP2-dependent invasion. In some embodiments, thecancer expresses or overexpresses NRP2 but does not substantiallyexpress neuropilin-1 (NRP1). Certain methods are directed to reducing orpreventing re-emergence of a cancer in a subject in need thereof,wherein administration of the therapeutic composition enables generationof an immune memory to the cancer. In some embodiments, the subject haslymphedema.

Certain embodiments comprise administering to the subject at least oneadditional agent selected from one or more of a cancer immunotherapyagent, a chemotherapeutic agent, a hormonal therapeutic agent, and akinase inhibitor. In some embodiments, the at least one anti-NRP2antibody or antigen-binding fragment thereof and the at least one agentare administered separately, as separate compositions. In someembodiments, the at least one anti-NRP2 antibody and the at least oneagent are administered together as part of the same therapeuticcomposition, optionally as a therapeutic composition described herein.In some embodiments, the cancer immunotherapy agent is selected from oneor more of an immune checkpoint modulatory agent, a cancer vaccine, anoncolytic virus, a cytokine, and a cell-based immunotherapies.

In some embodiments, the immune checkpoint modulatory agent is apolypeptide, optionally an antibody or antigen-binding fragment thereofor a ligand, or a small molecule. In some embodiments, the immunecheckpoint modulatory agent comprises

-   -   (a) an antagonist of a inhibitory immune checkpoint molecule; or    -   (b) an agonist of a stimulatory immune checkpoint molecule.    -   optionally, wherein the immune checkpoint modulatory agent        specifically binds to the immune checkpoint molecule.

In some embodiments, the inhibitory immune checkpoint molecule isselected from one or more of Programmed Death-Ligand 1 (PD-L1),Programmed Death 1 (PD-1), Programmed Death-Ligand 2 (PD-L2), CytotoxicT-Lymphocyte-Associated protein 4 (CTLA-4), Indoleamine 2,3-dioxygenase(IDO), tryptophan 2,3-dioxygenase (TDO), T-cell Immunoglobulin domainand Mucin domain 3 (TIM-3), Lymphocyte Activation Gene-3 (LAG-3),V-domain Ig suppressor of T cell activation (VISTA), B and T LymphocyteAttenuator (BTLA), CD160, Herpes Virus Entry Mediator (HVEM), and T-cellimmunoreceptor with Ig and ITIM domains (TIGIT).

In some embodiments:

-   -   the antagonist is a PD-L1 and/or PD-L2 antagonist optionally        selected from one or more of an antibody or antigen-binding        fragment or small molecule that specifically binds thereto,        atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab        (MEDI4736), optionally wherein the cancer is selected from one        or more of colorectal cancer, melanoma, breast cancer,        non-small-cell lung carcinoma, bladder cancer, and renal cell        carcinoma;    -   the antagonist is a PD-1 antagonist optionally selected from one        or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto, nivolumab,        pembrolizumab, MK-3475, AMP-224, AMP-514PDR001, and pidilizumab,        optionally wherein the PD-1 antagonist is nivolumab and the        cancer is optionally selected from one or more of Hodgkin's        lymphoma, melanoma, non-small cell lung cancer, hepatocellular        carcinoma, renal cell carcinoma, and ovarian cancer;    -   the PD-1 antagonist is pembrolizumab and the cancer is        optionally selected from one or more of melanoma, non-small cell        lung cancer, small cell lung cancer, head and neck cancer, and        urothelial cancer;    -   the antagonist is a CTLA-4 antagonist optionally selected from        one or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto, ipilimumab,        tremelimumab, optionally wherein the cancer is selected from one        or more of melanoma, prostate cancer, lung cancer, and bladder        cancer;    -   the antagonist is an IDO antagonist optionally selected from one        or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto, indoximod (NLG-8189),        1-methyl-tryptophan (1MT), β-Carboline (norharmane;        9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat, and        wherein the cancer is optionally selected from one or more of        metastatic breast cancer and brain cancer optionally        glioblastoma multiforme, glioma, gliosarcoma or malignant brain        tumor;    -   the antagonist is a TDO antagonist optionally selected from one        or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto, 680C91, and LM10;    -   the antagonist is a TIM-3 antagonist optionally selected from        one or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto;    -   the antagonist is a LAG-3 antagonist optionally selected from        one or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto, and BMS-986016;    -   the antagonist is a VISTA antagonist optionally selected from        one or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto;    -   the antagonist is a BTLA, CD160, and/or HVEM antagonist        optionally selected from one or more of an antibody or        antigen-binding fragment or small molecule that specifically        binds thereto;    -   the antagonist is a TIGIT antagonist optionally selected from        one or more of an antibody or antigen-binding fragment or small        molecule that specifically binds thereto.

In some embodiments, the stimulatory immune checkpoint molecule isselected from one or more of OX40, CD40, Glucocorticoid-Induced TNFRFamily Related Gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and HerpesVirus Entry Mediator (HVEM).

In some embodiments:

-   -   the agonist is an OX40 agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto, OX86,        Fc-OX40L, and GSK3174998;    -   the agonist is a CD40 agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto, CP-870,893,        dacetuzumab, Chi Lob 7/4, ADC-1013, and rhCD40L, and wherein the        cancer is optionally selected from one or more of melanoma,        pancreatic carcinoma, mesothelioma, and hematological cancers        optionally lymphoma such as Non-Hodgkin's lymphoma;    -   the agonist is a GITR agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto, INCAGN01876,        DTA-1, and MEDI1873;    -   the agonist is a CD137 agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto, utomilumab,        and 4-1BB ligand;    -   the agonist is a CD27 agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto, varlilumab,        and CDX-1127 (1F5);    -   the agonist is a CD28 agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto, and TAB08;        and/or    -   the agonist is an HVEM agonist optionally selected from one or        more of an antibody or antigen-binding fragment or small        molecule or ligand that specifically binds thereto.

In some embodiments, the cancer vaccine is selected from one or more ofOncophage, a human papillomavirus HPV vaccine optionally Gardasil orCervarix, a hepatitis B vaccine optionally Engerix-B, Recombivax HB, orTwinrix, and sipuleucel-T (Provenge), or comprises a cancer antigenselected from one or more of human Her2/neu, Her1/EGF receptor (EGFR),Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE Receptor),MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growthfactor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40,CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR,CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1(IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA),guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrinα5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblastactivation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (orCA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA),NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamilymember 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), and mesothelin, optionally wherein the subject has oris at risk for having a cancer that comprises the corresponding cancerantigen.

In some embodiments, the oncolytic virus selected from one or more oftalimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™),Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010),Senecavirus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102(Ad5/3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

In some embodiments, the cytokine selected from one or more ofinterferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and Granulocyte-macrophagecolony-stimulating factor (GM-CSF).

In some embodiments, the cell-based immunotherapy agent comprises cancerantigen-specific T-cells, optionally ex vivo-derived T-cells. In someembodiments, the cancer antigen-specific T-cells are selected from oneor more of chimeric antigen receptor (CAR)-modified T-cells, and T-cellReceptor (TCR)-modified T-cells, tumor infiltrating lymphocytes (TILs),and peptide-induced T-cells.

In some embodiments, the at least one chemotherapeutic agent is selectedfrom one or more of an alkylating agent, an anti-metabolite, a cytotoxicantibiotic, a topoisomerase inhibitor (type 1 or type II), and ananti-microtubule agent.

In some embodiments:

-   -   the alkylating agent is selected from one or more of nitrogen        mustards (optionally mechlorethamine, cyclophosphamide, mustine,        melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas        (optionally N-Nitroso-N-methylurea (MNU), carmustine (BCNU),        lomustine (CCNU), semustine (MeCCNU), fotemustine, and        streptozotocin), tetrazines (optionally dacarbazine,        mitozolomide, and temozolomide), aziridines (optionally        thiotepa, mytomycin, and diaziquone (AZQ)), cisplatins and        derivatives thereof (optionally carboplatin and oxaliplatin),        and non-classical alkylating agents (optionally procarbazine and        hexamethylmelamine);    -   the anti-metabolite is selected from one or more of anti-folates        (optionally methotrexate and pemetrexed), fluoropyrimidines        (optionally 5-fluorouracil and capecitabine), deoxynucleoside        analogues (optionally ancitabine, enocitabine, cytarabine,        gemcitabine, decitabine, azacitidine, fludarabine, nelarabine,        cladribine, clofarabine, fludarabine, and pentostatin), and        thiopurines (optionally thioguanine and mercaptopurine);    -   the cytotoxic antibiotic is selected from one or more of        anthracyclines (optionally doxorubicin, daunorubicin,        epirubicin, idarubicin, pirarubicin, aclarubicin, and        mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and        actinomycin;    -   the topoisomerase inhibitor is selected from one or more of        camptothecin, irinotecan, topotecan, etoposide, doxorubicin,        mitoxantrone, teniposide, novobiocin, merbarone, and        aclarubicin; and/or    -   the anti-microtubule agent is selected from one or more of        taxanes (optionally paclitaxel and docetaxel) and vinca        alkaloids (optionally vinblastine, vincristine, vindesine,        vinorelbine).

In some embodiments, the at least one hormonal therapeutic agent is ahormonal agonist or a hormonal antagonist. In some embodiments, thehormonal agonist is selected from one or more of a progestogen(progestin), a corticosteroid (optionally prednisolone,methylprednisolone, or dexamethasone), insulin like growth factors, VEGFderived angiogenic and lymphangiogenic factors (optionally VEGF-A,VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor(FGF), galectin, hepatocyte growth factor (HGF), platelet derived growthfactor (PDGF), transforming growth factor (TGF)-beta, an androgen, anestrogen, and a somatostatin analog. In some embodiments, the hormonalantagonist is selected from one or more of a hormone synthesisinhibitor, optionally an aromatase inhibitor or a gonadotropin-releasinghormone (GnRH) or an analog thereof, and a hormone receptor antagonist,optionally a selective estrogen receptor modulator (SERM) or ananti-androgen, or an antibody directed against a hormonal receptor,optionally cixutumumab, dalotuzumab, figitumumab, ganitumab,istiratumab, robatumumab, alacizumab pegol, bevacizumab, icrucumab,ramucirumab, fresolimumab, metelimumab, naxitamab, cetuximab,depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine,matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab,tomuzotuximab, zalutumumab, aprutumab ixadotin, bemarituzumab,olaratumab, or tovetumab.

In some embodiments, the kinase inhibitor is selected from one or moreof adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib,cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib,entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib,imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab,pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib,sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, andvemuafenib

In some embodiments, the cancer is a primary cancer. In someembodiments, the cancer is a metastatic cancer, optionally a metastaticcancer that expresses NRP2a and/or NRP2b. In some embodiments, thecancer is selected from one or more of melanoma (e.g., metastaticmelanoma), pancreatic cancer, bone cancer, prostate cancer, small celllung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia(e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloidleukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma(hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer,ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme,meningioma, pituitary adenoma, vestibular schwannoma, primary CNSlymphoma, primitive neuroectodermal tumor (medulloblastoma), kidneycancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer,esophageal cancer, brain cancer, head and neck cancers, cervical cancer,testicular cancer, thyroid cancer, and stomach cancer. In someembodiments, the metastatic cancer is selected from one or more of:

-   -   (a) a bladder cancer which has metastasized to the bone, liver,        and/or lungs;    -   (b) a breast cancer which has metastasized to the bone, brain,        liver, and/or lungs;    -   (c) a colorectal cancer which has metastasized to the liver,        lungs, and/or peritoneum;    -   (d) a kidney cancer which has metastasized to the adrenal        glands, bone, brain, liver, and/or lungs;    -   (e) a lung cancer which has metastasized to the adrenal glands,        bone, brain, liver, and/or other lung sites;    -   (f) a melanoma which has metastasized to the bone, brain, liver,        lung, and/or skin/muscle;    -   (g) a ovarian cancer which has metastasized to the liver, lung,        and/or peritoneum;    -   (h) a pancreatic cancer which has metastasized to the liver,        lung, and/or peritoneum;    -   (i) a prostate cancer which has metastasized to the adrenal        glands, bone, liver, and/or lungs;    -   (j) a stomach cancer which has metastasized to the liver, lung,        and/or peritoneum;    -   (l) a thyroid cancer which has metastasized to the bone, liver,        and/or lungs; and    -   (m) a uterine cancer which has metastasized to the bone, liver,        lung, peritoneum, and/or vagina.

In some embodiments, the subject has, and/or is selected for treatmentbased on having, increased circulating or serum levels of at least oneNRP2 ligand (optionally an NRP2 ligand from Table N2 or Table N3 and/oran HRS polypeptide from Table H1), either bound or free, relative to thelevels of a healthy control or matched control standard or population ofsubject(s), optionally about or at least about 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, or 5000 pM of theat least one NRP2 ligand, or about or at least about 30-100, 40-100,50-100, 30-2000, 40-2000, 50-2000, 60-2000, 70-2000, 80-2000, 90-2000,100-2000, 200-2000, 300-2000, 400-2000, 500-2000, 600-2000, 700-2000,800-2000, 900-2000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 pM ofthe at least one NRP2 ligand.

In some embodiments, the subject has, and/or is selected for treatmentbased on having, a disease associated with increased levels orexpression of at least one NRP2 ligand (optionally an NRP2 ligand fromTable N2 or Table N3 and/or an HRS polypeptide from Table H1) and/or acoding mRNA thereof relative to a healthy control or matched controlstandard or population of subject(s), optionally a cancer which hasincreased levels or expression of the at least one NRP2 ligand and/or acoding mRNA thereof relative to a non-cancerous control cell or tissue,optionally relative to a non-cancerous cell or tissue of the same typeas the cancer, optionally wherein the HRS polypeptide is a splicevariant selected from HisRS^(N2), HisRS^(N3), HisRS^(N4), HisRS^(N5),HisRS^(C1), HisRS^(C2), HisRS^(C3), HisRS^(C4), HisRS^(C5), HisRS^(C6),HisRS^(C7), HisRS^(C8), and HisRS^(C9).

In some embodiments, the subject has, and/or is selected for treatmentbased on having, increased circulating or serum levels of a solubleneuropilin 2 (NRP2) polypeptide (optionally selected from Table N1),either bound or free, relative to the levels of a healthy control ormatched control standard or population of subject(s), optionallycirculating or serum levels of about or at least about 10, 20, 30, 50,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000 pM of thesoluble NRP2 polypeptide, or optionally circulating or serum levelsabout 30-50, 50-100, 100-2000, 200-2000, 300-2000, 400-2000, 500-2000,600-2000, 700-2000, 800-2000, 900-2000, 1000-2000, 2000-3000, 3000-4000,4000-5000 pM of the soluble NRP2 polypeptide.

In some embodiments, the subject has, and/or is selected for treatmentbased on having, a disease associated with increased levels orexpression of an NRP2 polypeptide (optionally selected from Table N1)and/or a coding mRNA thereof relative to a healthy control or matchedcontrol standard or population of subject(s), optionally a cancer whichhas increased levels or expression of an NRP2 polypeptide (optionallyselected from Table N1) and/or a coding mRNA thereof relative to anon-cancerous control cell or tissue, optionally relative to anon-cancerous cell or tissue of the same type as the cancer.

In some embodiments, the subject has, and/or is selected for treatmentbased on having, a disease associated with increased levels orexpression of NRP2a and/or NRP2b, or an altered ratio of NRP2a:NRP2bexpression, relative to a healthy control or matched control standard orpopulation of subject(s). In some embodiments, the levels of NRP2b areincreased by about or at least about 10%, 20%, 30%, 40%, 50%, 100%,200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% compared to ahealthy control or matched control standard or population of subject(s).In some embodiments, the healthy control or matched control standard orpopulation of subject(s) comprises average ranges for age-matchedsamples of cancerous or non-cancerous cells or tissue of the same typeas the cancer, which comprise specific characteristics such as drugresistance, metastatic potential, aggressiveness, genetic signature(optionally p53 mutation(s), PTEN deletion, IGFR expression), and/orexpression patterns. In some embodiments, the subject has, and/or isselected for treatment based on having, increased circulating levels ofHRS:NRP2 complexes relative to a healthy or matched control standard orpopulation of subject(s).

Certain embodiments comprise administering the at least one anti-NRP2antibody in an amount and at a frequency sufficient to achieve anaverage, sustained serum or circulating levels of a soluble NRP2polypeptide of about or less than about 500 pM, 400 pM, 300 pM, 200 pM,100 pM, 50 pm, 40 pM, 30 pM, 20 pM, or 10 pM. Certain embodimentscomprise administering the at least one anti-NRP2 antibody in an amountand at a frequency sufficient to achieve a reduction in the circulatinglevels of HRS:NRP2 complexes, optionally a reduction of about or atleast about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95,99, or 100%.

In some embodiments, the at least one anti-NRP2 antibody enhances theimmune response to the cancer by about, or at least about, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000% or more, relative to a control. In someembodiments, the at least one anti-NRP2 antibody reduces the rate of invitro growth of the cancer by about or at least about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000% or more relative to an untreated control. In someembodiments, the at least one anti-NRP2 antibody reduces the in vitroadhesiveness of the cancer to a substrate by about or at least about, 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 2000% or more relative to an untreatedcontrol, optionally wherein the substrate comprises laminin.

In some embodiments, the at least one anti-NRP2 antibody reduces theinvasiveness of the cancer by about or at least about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000% or more relative to an untreated control. In someembodiments, the at least one anti-NRP2 antibody inhibits the rate ofmigration or motility of the cancer by about or at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000% or more relative to an untreatedcontrol. In some embodiments, the at least one anti-NRP2 antibodyinhibits the rate of autophagy or endosome maturation (optionallyendosome acidification) of the cancer or associated immune cells byabout or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or morerelative to an untreated control. In some embodiments, the at least oneanti-NRP2 antibody enhances the susceptibility of the cancer to anadditional agent selected from one or more of a chemotherapeutic agent,hormonal therapeutic agent, and kinase inhibitor by about or at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative to theadditional agent alone. In some embodiments, the at least one anti-NRP2antibody enhances an anti-tumor and/or immunostimulatory activity of thecancer immunotherapy agent by about, or at least about, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 2000% or more, relative to the cancer immunotherapyagent alone.

Certain embodiments comprise administering the at least one anti-NRP2antibody in an amount and at a frequency sufficient to achieve a steadystate concentration, or average circulating concentration, of the atleast one anti-NRP2 antibody of between about 1 nM and about 1 μM,between about 1 nM and about 100 nM, between about 1 nM and about 10 nM,or between about 1 nM and about 3 μM.

Also included are patient care kits, comprising:

-   -   (a) at least one antibody or antigen-binding fragment thereof        that specifically binds to a human neuropilin-2 (NRP2)        polypeptide; and optionally    -   (b) at least one additional agent selected from a cancer        immunotherapy agent, a chemotherapeutic agent, a hormonal        therapeutic agent, and a kinase inhibitor.

In some embodiments, (a) and (b) are in separate therapeuticcompositions. In some embodiments, (a) and (b) are in the sametherapeutic composition. In some embodiments, the at least onechemotherapeutic agent is selected from one or more of an alkylatingagent, an anti-metabolite, a cytotoxic antibiotic, a topoisomeraseinhibitor (type 1 or type II), and an anti-microtubule agent.

In some embodiments:

-   -   the alkylating agent is selected from one or more of nitrogen        mustards (optionally mechlorethamine, cyclophosphamide, mustine,        melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas        (optionally N-Nitroso-N-methylurea (MNU), carmustine (BCNU),        lomustine (CCNU), semustine (MeCCNU), fotemustine, and        streptozotocin), tetrazines (optionally dacarbazine,        mitozolomide, and temozolomide), aziridines (optionally        thiotepa, mytomycin, and diaziquone (AZQ)), cisplatins and        derivatives thereof (optionally carboplatin and oxaliplatin),        and non-classical alkylating agents (optionally procarbazine and        hexamethylmelamine);    -   the anti-metabolite is selected from one or more of anti-folates        (optionally methotrexate and pemetrexed), fluoropyrimidines        (optionally 5-fluorouracil and capecitabine), deoxynucleoside        analogues (optionally ancitabine, enocitabine, cytarabine,        gemcitabine, decitabine, azacitidine, fludarabine, nelarabine,        cladribine, clofarabine, fludarabine, and pentostatin), and        thiopurines (optionally thioguanine and mercaptopurine);    -   the cytotoxic antibiotic is selected from one or more of        anthracyclines (optionally doxorubicin, daunorubicin,        epirubicin, idarubicin, pirarubicin, aclarubicin, and        mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and        actinomycin;    -   the topoisomerase inhibitor is selected from one or more of        camptothecin, irinotecan, topotecan, etoposide, doxorubicin,        mitoxantrone, teniposide, novobiocin, merbarone, and        aclarubicin; and/or    -   the anti-microtubule agent is selected from one or more of        taxanes (optionally paclitaxel and docetaxel) and vinca        alkaloids (optionally vinblastine, vincristine, vindesine,        vinorelbine).

In some embodiments, the at least one hormonal therapeutic agent is ahormonal agonist or a hormonal antagonist. In some embodiments, thehormonal agonist is selected from one or more of a progestogen(progestin), a corticosteroid (optionally prednisolone,methylprednisolone, or dexamethasone), insulin like growth factors, VEGFderived angiogenic and lymphangiogenic factors (optionally VEGF-A,VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor(FGF), galectin, hepatocyte growth factor (HGF), platelet derived growthfactor (PDGF), transforming growth factor (TGF)-beta, an androgen, anestrogen, and a somatostatin analog. In some embodiments, the hormonalantagonist is selected from one or more of a hormone synthesisinhibitor, optionally an aromatase inhibitor or a gonadotropin-releasinghormone (GnRH) or an analog thereof, and a hormone receptor antagonist,optionally a selective estrogen receptor modulator (SERM) or ananti-androgen, or an antibody directed against a hormonal receptor,optionally cixutumumab, dalotuzumab, figitumumab, ganitumab,istiratumab, robatumumab, alacizumab pegol, bevacizumab, icrucumab,ramucirumab, fresolimumab, metelimumab, naxitamab, cetuximab,depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine,matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab,tomuzotuximab, zalutumumab, aprutumab ixadotin, bemarituzumab,olaratumab, or tovetumab.

In some embodiments, the kinase inhibitor is selected from one or moreof adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib,cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib,entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib,imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab,pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib,sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, andvemuafenib.

Also included are bioassay systems, comprising a substantially pureanti-NRP2 antibody or antigen-binding fragment thereof, optionally asdefined herein, and a host cell line that expresses a human NRP2polypeptide on the cell surface. In some embodiments, the NRP2polypeptide is labeled with a detectable label. In some embodiments, theanti-NRP2 antibody is labeled with a detectable label. In someembodiments, the NRP2 polypeptide is functionally coupled to a readoutor indicator, such as a fluorescent or luminescent indicator ofbiological activity of the NRP2 polypeptide. In some embodiments, theNRP2 polypeptide is selected from Table N1. Certain bioassay systemscomprise at least one NRP2 ligand (optionally an NRP2 ligand selectedfrom Table N2 or Table N3 and/or a human histidyl-tRNA synthetase (HRS)polypeptide selected from Table H1), optionally wherein the host cellexpresses the at least one NRP2 ligand. In some embodiments, the HRSpolypeptide is selected from Table H1, optionally wherein the HRSpolypeptide comprises a HRS splice variant, optionally selected fromHisRS^(N1), HisRS^(N3), HisRS^(N4), HisRS^(N5), HisRS^(C1), HisRS^(C2),HisRS^(C3), HisRS^(C4), HisRS^(C5), HisRS^(C6), HisRS^(C6), HisRS^(C7),HisRS^(C8), and HisRS^(C9). In some embodiments, the at least one NRP2ligand is selected from Table N2 or Table N3.

Also included are detection systems, comprising a cell that expresses ahuman neuropilin 2 (NRP2) polypeptide, at least one NRP2 ligand(optionally a recombinant NRP2 ligand selected from Table N2 or Table N3and/or a human histidyl-tRNA synthetase (HRS) polypeptide selected fromTable H1), and a human or humanized anti-NRP2 antibody orantigen-binding fragment thereof, optionally as defined herein, whichmodulates the interaction between the NRP2 polypeptide and the at leastone NRP2 ligand. In some embodiments, the anti-NRP2 antibody is labeledwith a detectable label. In some embodiments, the NRP2 polypeptide isselected from Table N1. In some embodiments, the HRS polypeptidecomprises a HRS splice variant selected from Table H1, optionallyselected from HisRS^(N1), HisRS^(N2), HisRS^(N3), HisRS^(N4),HisRS^(N5), HisRS^(C1), HisRS^(C2), HisRS^(C3), HisRS^(C4), HisRS^(C5),HisRS^(C6), HisRS^(C7), HisRS^(C8), and HisRS^(C9). In some embodiments,the at least one NRP2 ligand is selected from Table N2 or Table N3. Insome embodiments, the NRP2 polypeptide and/or the at least one NRP2ligand is/are functionally coupled to a readout or indicator, such as afluorescent or luminescent indicator of biological activity of the NRP2polypeptide or the at least one NRP2 ligand.

Also included are diagnostic systems, comprising a cell that comprises aneuropilin 2 (NRP2) polypeptide, and at least one NRP2 ligand thatspecifically binds to the NRP2 polypeptide (optionally an NRP2 ligandselected from Table N2 or Table N3 and/or a human histidyl-tRNAsynthetase (HRS) polypeptide selected from Table H1), wherein the cellcomprises an indicator molecule that indicates a change in the levels oractivity of the NRP2 polypeptide in response to interaction with the atleast one NRP2 ligand.

Also included are cellular compositions, comprising an engineeredpopulation of cells in which at least one cell comprises one or morepolynucleotides encoding a human or humanized anti-NRP2 antibody orantigen-binding fragment thereof, as defined herein, wherein the cellsare capable of growing in a serum-free medium.

Also included are cellular growth devices, comprising a human orhumanized anti-NRP2 antibody or antigen-binding fragment thereof, asdefined herein, an engineered population of cells in which at least onecell comprises one or more polynucleotides encoding said anti-NRP2antibody or antigen-binding fragment thereof, at least about 10 litersof a serum-free growth medium, and a sterile container.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate the general domain structure of neuropilins (1A)and exemplary neuropilin co-receptor functions (1B).

FIGS. 2A-2B illustrate the domain structure of NRP2 isoforms andexemplary NRP2 ligand binding domains. All NRP2 isoforms are identicalthrough the MAM domain, after this, is a short possible retained intronthat codes for GENFK. There are then two possible splicing frames (formsa and b), these code different juxtamembrane domains, transmembranehelixes, and cytoplasmic domains. Within the first there is analternative splice acceptor that removes 17 amino acids (variant 3, orform c). Each form has different spacing from the membrane, which mayaffect co-receptor specificity. The A/C and B forms, also have differenttransmembrane domains, where the A/C forms contain a dimerization motif(GXXXG).

FIG. 3 shows the binding of anti-NRP2 antibodies to clonal Expi293 cellsexpressing human NRP2. The four anti-NRP2 antibodies, and isotypecontrol antibody (human IgG4) were added to human NRP2 expressing cellsat the concentrations shown, and cell binding determined by FACSanalysis as described in the examples.

FIG. 4 shows the binding of anti-NRP2 antibodies to clonal Expi293 cellsexpressing cynomolgus monkey NRP2. The four anti-NRP2 antibodies, andisotype control antibody (human IgG4) were added to cynomologus monkeyNRP2 expressing cells at the concentrations shown, and cell bindingdetermined by FACS analysis as described in the examples.

FIGS. 5A-5B show the effects of anti-NRP2 antibodies on NRP2 receptordimerization with KDR in the presence and absence of VEGF-A. Theindicated antibodies were assessed in a receptor dimerization assay asdescribed in the examples. 5A shows normalized responses for eachindividual well were calculated to the time point prior to addition ofligand, then normalized to no-antibody/no-ligand (baseline signal).Replicates were then processed to give average response and standarddeviation. 5B shows the net effect at 80 minutes

FIGS. 6A-6B show the effects of anti-NRP2 antibodies on NRP2 receptordimerization with FLT4 in the presence and absence of VEGF-C. Theindicated antibodies were assessed in a receptor dimerization assay asdescribed in the examples. 6A shows normalized responses for eachindividual well were calculated to the time point prior to addition ofligand, then normalized to no-antibody/no-ligand (baseline signal).Replicates were then processed to give average response and standarddeviation. 6B shows the net effect at 80 minutes

FIGS. 7A-7B show the effects of anti-NRP2 antibodies on NRP2 receptordimerization with PLXNA1 in the presence and absence of SEMA 3F. Theindicated antibodies were assessed in a receptor dimerization assay asdescribed in the examples. 7A shows normalized responses for eachindividual well were calculated to the time point prior to addition ofligand, then normalized to no-antibody/no-ligand (baseline signal).Replicates were then processed to give average response and standarddeviation. 7B shows the net effect at 80 minutes

FIGS. 8A-8B show the effects of anti-NRP2 antibodies on NRP2 receptordimerization with KDR in the presence and absence of VEGF-A. Theindicated antibodies were assessed in a receptor dimerization assay asdescribed in the examples. 8A shows normalized responses for eachindividual well were calculated to the time point prior to addition ofligand, then normalized to no-antibody/no-ligand (baseline signal).Replicates were then processed to give average response and standarddeviation. 8B shows the net effect at 80 minutes.

FIGS. 9A-9B show the effects of anti-NRP2 antibodies on NRP2 receptordimerization with FLT4 in the presence and absence of VEGF-C. Theindicated antibodies were assessed in a receptor dimerization assay asdescribed in the examples. 9A shows normalized responses for eachindividual well were calculated to the time point prior to addition ofligand, then normalized to no-antibody/no-ligand (baseline signal).Replicates were then processed to give average response and standarddeviation. 9B shows the net effect at 80 minutes.

FIGS. 10A-10B show the effects of anti-NRP2 antibodies on NRP2 receptordimerization with PLXN A1 in the presence and absence of SEMA 3F. Theindicated antibodies were assessed in a receptor dimerization assay asdescribed in the examples. 10A shows normalized responses for eachindividual well were calculated to the time point prior to addition ofligand, then normalized to no-antibody/no-ligand (baseline signal).Replicates were then processed to give average response and standarddeviation. 10B shows the net effect at 80 minutes

FIGS. 11A-11B show the measurement of antibody binding to Expi293 cellsover-expressing NRP2. 11A shows an example of MFI profiles stained withmouse/human reactive control antibody where untransfected cell histogramis shown in light grey, while cells over-expressing mouse NRP2 I383Vvariant are shown in dark grey. Gating for NRP2 over-expressing cells isshown. 11B shows a chart of MFI staining of aNRP2-10v10 at 10 nM ofhuman and mouse wild type NRP2, and mouse NRP2 receptors containingindividual mutations representing human residues.

FIG. 12 shows the structure of the human NRP2 b1 domain from PBD file2QQK. A ribbon diagram showing amino acids 280-426 is displayed. Sidechains of residues 299Y, 354N, 416S, and 319T are highlighted in white.Distances between the alpha carbon of 299Y, 354N, and 416S in angstromsare shown alongside a dotted line.

FIGS. 13A-13B shows the inhibitory effect of the anti-NRP2 antibody onanchorage-independent growth and sensitivity to chemotherapeutic agentsin triple negative breast cancer cells (TNBC) cells in 3D soft agarcolony formation assays. The fluorescence readings of the colonyformation assay are shown as dot plots with mean SEM, for TNBC cellsMDA-MB-231 (13A) or BT549 (13B) treated with a-NRP2-10v10 vs the isotypecontrol hIgG4 at 100 nM in combination with chemo drugs—Cisplatin or5-FU. The statistical significance is indicated by asterisks (*<0.05,**<0.01, ****<0.0001 by Student's t test). In both cases, the anti-tumoreffects of the anti-NRP2 antibody are significantly more pronounced incombination with cisplatin or 5-FU.

FIG. 14 shows the inhibitory effect of the anti-NRP2 antibody incombination with chemotherapeutic agents and a VEGF-A antibody drug(Bevacizumab) on anchorage-independent growth of TNBC MDA-MB-231 cellsin 3D methylcellulose colony formation assays. The luminescence readingsof the colony formation assay are shown as dot plots with mean SEM, forTNBC cells MDA-MB-231 treated with aNRP2-10v10 vs the isotype controlhIgG4 at 100 nM in combination with chemo drugs—Cisplatin (at 2 doses)or Bevacizumab (at 100 nM). The statistical significance is indicated byasterisks (**p<0.01, ***p<0.001, ****p<0.0001 by Student's t test).

FIG. 15 shows the inhibition of human lymphatic endothelial cell (HLEC)migration toward vascular endothelial growth factor C (VEGF-C) byantibodies aNRP2-10v10 and aNRP2-11v7. HLECs were inhibited by both NRP2antibodies to levels consistent with positive control antibodies (αKDRor 3C5) directed against VEGFR2 or VEGFR3 respectively.

FIG. 16 shows that treatment of cells with the antibodies aNRP2-14v10(NRP2_14) or aNRP2-11v7 (NRP2_11) blocked the reduction in phospho-Aktlevels upon Semaphorin 3F treatment in U251 glioblastoma cells.Treatment of U251 cells with Sema3F results in an approximately 50%decrease in intracellular phospho-Akt levels that can be blocked by bothNRP2_11 and NRP2_14, but is not blocked by VEGF-C blocking antibodyaNRP2-10v10 or by mIgG1 and hIgG4 control antibodies.

FIG. 17 show a schematic of Sema3F signaling via NRP2. Treatment withSema3F results in blocking PI3K activity and thereby reducingphospho-Akt levels.

FIG. 18 shows that antibodies aNRP2-14v10 and aNRP22-11v7, but notaNRP2-10v10, block semaphorin 3F mediated inhibition of AKTphosphorylation. Treatment of U251 cells with Sema3F resulted in anapproximately 50% decrease in intracellular phospho-Akt levels that canbe blocked by both the semaphorin blocking antibodies aNRP2-11v7 andaNRP2-14v10, but is not blocked by the VEGF-C blocking antibodyaNRP2-10v10 or with mIgG1 and hIgG4 control antibodies.

FIGS. 19A-19B show that the mouse surrogate VEGF Blocking antibodyaNRP2-28 inhibits Tumor Growth in a Murine Melanoma Model (B16.F10).Animals bearing B16-F10 tumors and treated with three doses of the mousesurrogate antibody aNRP2-28, which recognizes mouse NRP2 (and is afunctional surrogate for anti-human NRP2 antibody aNRP2-10v10). FIG. 19Ashows that treatment with the mouse surrogate antibody aNRP2-28 (greytriangles), showed tumor growth inhibition compared to the IgG controlgroup (black squares), and Bevacizumab (black triangles) reachingstatistical significance (p<0.05) on Day 14, 16, and 19. FIG. 19B showsterminal tumor weights for each treatment group.

FIG. 20A-20B show the anti-metastatic effects of VEGF-blocker (aNRP2-28)in a model of spontaneous metastasis (4T1). FIG. 20A shows that animalsbearing 4T1 tumors and treated with mouse surrogate antibody aNRP2-28showed a reduction in metastatic nodules in the lung at termination,reaching statistical significance (p≤0.05) compared to the IgG control(black bar graph). FIG. 20B shows serum antibody concentration versusthe lung of lung metastases, demonstrating a linear correlation betweenterminal antibody concentration and number of metastatic nodules

FIG. 21 shows the synergistic effect of VEGF-Blockers(aNRP2-10v10/aNRP2-28) in combination with cisplatin in the TNBCXenograft Model (MDA-MB-231). The addition of pooled anti-NRP2antibodies (black symbols) to the cisplatin treatment regimen increasedthe tumor-inhibitory effect of cisplatin starting on day 40 postinoculation, reaching statistical significance on day 60 compared to thecontrol animals (grey symbols).

FIG. 22 shows that the VEGF Blocker (aNRP2-10v10) enhances activity ofthe chemotherapeutic drug 5-FU in the NSCLC Xenograft model (A549). Thecombination of 5-FU and aNRP2-10 (black symbols) performed better than5-FU with control IgG (grey symbols), evidencing that targeting NRP2increases the efficacy of the chemotherapeutic drug 5-FU in anart-accepted model of NSCLC. A statistically significant difference wasreached on day 51 post cell inoculation

FIGS. 23A-23B show that the VEGF Blocker (aNRP2-28) inhibitsneo-lymphangiogenesis in an animal model of corneal injury. FIG. 23Ashows representative image scans of Lyve-1 (lymphangiogenic marker)stained corneas of the IgG control and aNRP2-28 group. Plotting the meanvalues of % Lye-1* area for each treatment group. FIG. 23B shows a cleartrend towards reduced sprouting of lymphatic vessels in the aNRP2-10treatment groups as compared to the IgG control group.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the disclosure belongs. Although any methods,materials, compositions, reagents, cells, similar or equivalent similaror equivalent to those described herein can be used in the practice ortesting of the subject matter of the present disclosure, preferredmethods and materials are described. All publications and references,including but not limited to patents and patent applications, cited inthis specification are herein incorporated by reference in theirentirety as if each individual publication or reference werespecifically and individually indicated to be incorporated by referenceherein as being fully set forth. Any patent application to which thisapplication claims priority is also incorporated by reference herein inits entirety in the manner described above for publications andreferences.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. These and relatedtechniques and procedures may be generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification. Unless specific definitions areprovided, the nomenclature utilized in connection with, and thelaboratory procedures and techniques of, molecular biology, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well-known and commonly used in theart. Standard techniques may be used for recombinant technology,molecular biological, microbiological, chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

For the purposes of the present disclosure, the following terms aredefined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” includes “one element”, “one ormore elements” and/or “at least one element”.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes. As used herein, the term “antigen”includes substances that are capable, under appropriate conditions, ofinducing an immune response to the substance and of reacting with theproducts of the immune response. For example, an antigen can berecognized by antibodies (humoral immune response) or sensitizedT-lymphocytes (T helper or cell-mediated immune response), or both.Antigens can be soluble substances, such as toxins and foreign proteins,or particulates, such as bacteria and tissue cells; however, only theportion of the protein or polysaccharide molecule known as the antigenicdeterminant (epitopes) combines with the antibody or a specific receptoron a lymphocyte. More broadly, the term “antigen” includes any substanceto which an antibody binds, or for which antibodies are desired,regardless of whether the substance is immunogenic. For such antigens,antibodies can be identified by recombinant methods, independently ofany immune response.

An “antagonist” refers to biological structure or chemical agent thatinterferes with or otherwise reduces the physiological action of anotheragent or molecule. In some instances, the antagonist specifically bindsto the other agent or molecule. Included are full and partialantagonists.

An “agonist” refers to biological structure or chemical agent thatincreases or enhances the physiological action of another agent ormolecule. In some instances, the agonist specifically binds to the otheragent or molecule. Included are full and partial agonists.

The term “anergy” refers to the functional inactivation of a T-cell, orB-cell response to re-stimulation by antigen.

As used herein, the term “amino acid” is intended to mean both naturallyoccurring and non-naturally occurring amino acids as well as amino acidanalogs and mimetics. Naturally-occurring amino acids include the 20(L)-amino acids utilized during protein biosynthesis as well as otherssuch as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine,homocysteine, citrulline and ornithine, for example. Non-naturallyoccurring amino acids include, for example, (D)-amino acids, norleucine,norvaline, p-fluorophenylalanine, ethionine and the like, which areknown to a person skilled in the art. Amino acid analogs includemodified forms of naturally and non-naturally occurring amino acids.Such modifications can include, for example, substitution or replacementof chemical groups and moieties on the amino acid or by derivatizationof the amino acid. Amino acid mimetics include, for example, organicstructures which exhibit functionally similar properties such as chargeand charge spacing characteristic of the reference amino acid. Forexample, an organic structure which mimics arginine (Arg or R) wouldhave a positive charge moiety located in similar molecular space andhaving the same degree of mobility as the e-amino group of the sidechain of the naturally occurring Arg amino acid. Mimetics also includeconstrained structures so as to maintain optimal spacing and chargeinteractions of the amino acid or of the amino acid functional groups.Those skilled in the art know or can determine what structuresconstitute functionally equivalent amino acid analogs and amino acidmimetics.

As used herein, the term “antibody” encompasses not only intactpolyclonal or monoclonal antibodies, but also fragments thereof (such asdAb, Fab, Fab′, F(ab′)2, Fv), single chain (ScFv), synthetic variantsthereof, naturally occurring variants, fusion proteins comprising anantibody portion with an antigen-binding fragment of the requiredspecificity, humanized antibodies, chimeric antibodies, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen-binding site or fragment (epitope recognition site) of therequired specificity. Certain features and characteristics of antibodies(and antigen-binding fragments thereof) are described in greater detailherein.

An antibody or antigen-binding fragment can be of essentially any type.As is well known in the art, an antibody is an immunoglobulin moleculecapable of specific binding to a target, such as an immune checkpointmolecule, through at least one epitope recognition site, located in thevariable region of the immunoglobulin molecule.

The term “antigen-binding fragment” as used herein refers to apolypeptide fragment that contains at least one CDR of an immunoglobulinheavy and/or light chain that binds to the antigen of interest. In thisregard, an antigen-binding fragment of the herein described antibodiesmay comprise 1, 2, 3, 4, 5, or all 6 CDRs of a V_(H) and V_(L) sequencefrom antibodies that bind to a target molecule.

The binding properties of antibodies and antigen-binding fragmentsthereof can be quantified using methods well known in the art (seeDavies et al., Annual Rev. Biochem. 59:439-473, 1990). In someembodiments, an antibody or antigen-binding fragment thereofspecifically binds to a target molecule, for example, an NRP2polypeptide or an epitope or complex thereof, with an equilibriumdissociation constant that is about or ranges from about ≤10⁻⁷ M toabout 10⁻⁸ M. In some embodiments, the equilibrium dissociation constantis about or ranges from about ≤10⁻⁹ M to about ≤10⁻¹ M. In certainillustrative embodiments, an antibody or antigen-binding fragmentthereof has an affinity (Kd or EC₅₀) for a target molecule (to which itspecifically binds) of about, at least about, or less than about, 0.01,0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 40, or 50 nM.

A molecule such as a polypeptide or antibody is said to exhibit“specific binding” or “preferential binding” if it reacts or associatesmore frequently, more rapidly, with greater duration and/or with greateraffinity with a particular cell, substance, or particular epitope thanit does with alternative cells or substances, or epitopes. An antibody“specifically binds” or “preferentially binds” to a target molecule orepitope if it binds with greater affinity, avidity, more readily, and/orwith greater duration than it binds to other substances or epitopes, forexample, by a statistically significant amount. Typically one member ofthe pair of molecules that exhibit specific binding has an area on itssurface, or a cavity, which specifically binds to and is thereforecomplementary to a particular spatial and/or polar organization of theother member of the pair of molecules. Thus, the members of the pairhave the property of binding specifically to each other. For instance,an antibody that specifically or preferentially binds to a specificepitope is an antibody that binds that specific epitope with greateraffinity, avidity, more readily, and/or with greater duration than itbinds to other epitopes. It is also understood by reading thisdefinition that, for example, an antibody (or moiety or epitope) thatspecifically or preferentially binds to a first target may or may notspecifically or preferentially bind to a second target. The term is alsoapplicable where, for example, an antibody is specific for a particularepitope which is carried by a number of antigens, in which case thespecific binding member carrying the antigen-binding fragment or domainwill be able to bind to the various antigens carrying the epitope; forexample, it may be cross reactive to a number of different forms of atarget antigen from multiple species that share a common epitope

Immunological binding generally refers to the non-covalent interactionsof the type which occur between an immunoglobulin molecule and anantigen for which the immunoglobulin is specific, for example by way ofillustration and not limitation, as a result of electrostatic, ionic,hydrophilic and/or hydrophobic attractions or repulsion, steric forces,hydrogen bonding, van der Waals forces, and other interactions. Thestrength, or affinity of immunological binding interactions can beexpressed in terms of the dissociation constant (Kd) of the interaction,wherein a smaller Kd represents a greater affinity. Immunologicalbinding properties of selected polypeptides can be quantified usingmethods well known in the art. One such method entails measuring therates of antigen-binding site/antigen complex formation anddissociation, wherein those rates depend on the concentrations of thecomplex partners, the affinity of the interaction, and on geometricparameters that equally influence the rate in both directions. Thus,both the “on rate constant” (Kon) and the “off rate constant” (Koff) canbe determined by calculation of the concentrations and the actual ratesof association and dissociation. The ratio of Koff/Kon enablescancellation of all parameters not related to affinity, and is thusequal to the dissociation constant Kd. As used herein, the term“affinity” includes the equilibrium constant for the reversible bindingof two agents and is expressed as Kd or EC₅₀. Affinity of a bindingprotein to a ligand such as affinity of an antibody for an epitope canbe, for example, from about 100 nanomolar (nM) to about 0.1 nM, fromabout 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1femtomolar (fM). As used herein, the term “avidity” refers to theresistance of a complex of two or more agents to dissociation afterdilution. In some embodiments, affinity is expressed in the terms of thehalf maximal effective concentration (EC₅₀), which refers to theconcentration of an agent, such as an antibody, or an anti-NRP2antibody, as disclosed herein, which induces a response halfway betweenthe baseline and maximum after a specified exposure time. The EC₅₀ iscommonly used as a measure of an antibody's potency.

Antibodies may be prepared by any of a variety of techniques known tothose of ordinary skill in the art. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.Monoclonal antibodies specific for a polypeptide of interest may beprepared, for example, using the technique of Kohler and Milstein, Eur.J. Immunol. 6:511-519, 1976, and improvements thereto. Also included aremethods that utilize transgenic animals such as mice to express humanantibodies. See, e.g., Neuberger et al., Nature Biotechnology 14:826,1996; Lonberg et al., Handbook of Experimental Pharmacology 113:49-101,1994; and Lonberg et al., Internal Review of Immunology 13:65-93, 1995.Particular examples include the VELOCIMMUNE® platform by REGENEREX®(see, e.g., U.S. Pat. No. 6,596,541).

Antibodies can also be generated or identified by the use of phagedisplay or yeast display libraries (see, e.g., U.S. Pat. No. 7,244,592;Chao et al., Nature Protocols. 1:755-768, 2006). Non-limiting examplesof available libraries include cloned or synthetic libraries, such asthe Human Combinatorial Antibody Library (HuCAL), in which thestructural diversity of the human antibody repertoire is represented byseven heavy chain and seven light chain variable region genes. Thecombination of these genes gives rise to 49 frameworks in the masterlibrary. By superimposing highly variable genetic cassettes(CDRs=complementarity determining regions) on these frameworks, the vasthuman antibody repertoire can be reproduced. Also included are humanlibraries designed with human-donor-sourced fragments encoding alight-chain variable region, a heavy-chain CDR-3, synthetic DNA encodingdiversity in heavy-chain CDR-1, and synthetic DNA encoding diversity inheavy-chain CDR-2. Other libraries suitable for use will be apparent topersons skilled in the art.

In certain embodiments, antibodies and antigen-binding fragments thereofas described herein include a heavy chain and a light chain CDR set,respectively interposed between a heavy chain and a light chainframework region (FR) set which provide support to the CDRs and definethe spatial relationship of the CDRs relative to each other. As usedherein, the term “CDR set” refers to the three hypervariable regions ofa heavy or light chain V region. Proceeding from the N-terminus of aheavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and“CDR3” respectively. An antigen-binding site, therefore, includes sixCDRs, comprising the CDR set from each of a heavy and a light chain Vregion. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 orCDR3) is referred to herein as a “molecular recognition unit.”Crystallographic analysis of a number of antigen-antibody complexes hasdemonstrated that the amino acid residues of CDRs form extensive contactwith bound antigen, wherein the most extensive antigen contact is withthe heavy chain CDR3. Thus, the molecular recognition units areprimarily responsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRs. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

The structures and locations of immunoglobulin variable domains may bedetermined by reference to Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest. 4th Edition. US Department of Health and HumanServices. 1987, and updates thereof.

Also include are “monoclonal” antibodies, which refer to a homogeneousantibody population wherein the monoclonal antibody is comprised ofamino acids (naturally occurring and non-naturally occurring) that areinvolved in the selective binding of an epitope. Monoclonal antibodiesare highly specific, being directed against a single epitope. The term“monoclonal antibody” encompasses not only intact monoclonal antibodiesand full-length monoclonal antibodies, but also fragments thereof (suchas Fab, Fab′, F(ab′)2, Fv), single chain (ScFv), variants thereof,fusion proteins comprising an antigen-binding portion, humanizedmonoclonal antibodies, chimeric monoclonal antibodies, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen-binding fragment (epitope recognition site) of the requiredspecificity and the ability to bind to an epitope. It is not intended tobe limited as regards the source of the antibody or the manner in whichit is made (e.g., by hybridoma, phage selection, recombinant expression,transgenic animals). The term includes whole immunoglobulins as well asthe fragments etc. described above under the definition of “antibody.”

The proteolytic enzyme papain preferentially cleaves IgG molecules toyield several fragments, two of which (the F(ab) fragments) eachcomprise a covalent heterodimer that includes an intact antigen-bindingsite. The enzyme pepsin is able to cleave IgG molecules to provideseveral fragments, including the F(ab′)2 fragment which comprises bothantigen-binding sites. An Fv fragment for use according to certainembodiments can be produced by preferential proteolytic cleavage of anIgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fvfragments are, however, more commonly derived using recombinanttechniques known in the art. The Fv fragment includes a non-covalentVH::VL heterodimer including an antigen-binding site which retains muchof the antigen recognition and binding capabilities of the nativeantibody molecule. See Inbar et al., PNAS USA. 69:2659-2662, 1972;Hochman et al., Biochem. 15:2706-2710, 1976; and Ehrlich et al.,Biochem. 19:4091-4096, 1980.

In certain embodiments, single chain Fv (scFV) antibodies arecontemplated. For example, Kappa bodies (Ill et al., Prot. Eng.10:949-57, 1997); minibodies (Martin et al., EMBO J 13:5305-9, 1994);diabodies (Holliger et al., PNAS 90: 6444-8, 1993); or Janusins(Traunecker et al., EMBO J 10: 3655-59, 1991; and Traunecker et al.,Int. J. Cancer Suppl. 7:51-52, 1992), may be prepared using standardmolecular biology techniques following the teachings of the presentapplication with regard to selecting antibodies having the desiredspecificity.

A single chain Fv (scFv) polypeptide is a covalently linked VH::VLheterodimer which is expressed from a gene fusion including VH- andVL-encoding genes linked by a peptide-encoding linker. Huston et al.(PNAS USA. 85(16):5879-5883, 1988). A number of methods have beendescribed to discern chemical structures for converting the naturallyaggregated—but chemically separated-light and heavy polypeptide chainsfrom an antibody V region into an scFv molecule which will fold into athree dimensional structure substantially similar to the structure of anantigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405,to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al.

In certain embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of a “diabody.” Diabodies are multimersof polypeptides, each polypeptide comprising a first domain comprising abinding region of an immunoglobulin light chain and a second domaincomprising a binding region of an immunoglobulin heavy chain, the twodomains being linked (e.g., by a peptide linker) but unable to associatewith each other to form an antigen-binding site: antigen-binding sitesare formed by the association of the first domain of one polypeptidewithin the multimer with the second domain of another polypeptide withinthe multimer (WO94/13804). A dAb fragment of an antibody consists of aVH domain (Ward et al., Nature 341:544-546, 1989). Diabodies and othermultivalent or multispecific fragments can be constructed, for example,by gene fusion (see WO94/13804; and Holliger et al., PNAS USA.90:6444-6448, 1993)).

Minibodies comprising a scFvjoined to a CH3 domain are also included(see Hu et al., Cancer Res. 56:3055-3061, 1996). See also Ward et al.,Nature. 341:544-546, 1989; Bird et al., Science. 242:423-426, 1988;Huston et al., PNAS USA. 85:5879-5883, 1988); PCT/US92/09965;WO94/13804; and Reiter et al., Nature Biotech. 14:1239-1245, 1996.

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993),e.g., prepared chemically or from hybrid hybridomas, or may be any ofthe bispecific antibody fragments mentioned above. Diabodies and scFvcan be constructed without an Fc region, using only variable domains,potentially reducing the effects of anti-idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against antigen X, then a library can be made where the otherarm is varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by knobs-into-holes engineering(Ridgeway et al., Protein Eng., 9:616-621, 1996).

In certain embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of a UniBody®. A UniBody® is an IgG4antibody with the hinge region removed (see GenMab Utrecht, TheNetherlands; see also, e.g., US20090226421). This antibody technologycreates a stable, smaller antibody format with an anticipated longertherapeutic window than current small antibody formats. IgG4 antibodiesare considered inert and thus do not interact with the immune system.Fully human IgG4 antibodies may be modified by eliminating the hingeregion of the antibody to obtain half-molecule fragments having distinctstability properties relative to the corresponding intact IgG4 (GenMab,Utrecht). Halving the IgG4 molecule leaves only one area on the UniBody®that can bind to cognate antigens (e.g., disease targets) and theUniBody® therefore binds univalently to only one site on target cells.For certain cancer cell surface antigens, this univalent binding may notstimulate the cancer cells to grow as may be seen using bivalentantibodies having the same antigen specificity, and hence UniBody®technology may afford treatment options for some types of cancer thatmay be refractory to treatment with conventional antibodies. The smallsize of the UniBody® can be a great benefit when treating some forms ofcancer, allowing for better distribution of the molecule over largersolid tumors and potentially increasing efficacy.

In certain embodiments, the antibodies and antigen-binding fragmentsdescribed herein are in the form of a nanobody. Minibodies are encodedby single genes and are efficiently produced in almost all prokaryoticand eukaryotic hosts, for example, E. coli (see U.S. Pat. No.6,765,087), molds (for example Aspergillus or Trichoderma) and yeast(for example Saccharomyces, Kluyvermyces, Hansenula or Pichia (see U.S.Pat. No. 6,838,254). The production process is scalable andmulti-kilogram quantities of nanobodies have been produced. Nanobodiesmay be formulated as a ready-to-use solution having a long shelf life.The Nanoclone method (see WO 06/079372) is a proprietary method forgenerating Nanobodies against a desired target, based on automatedhigh-throughput selection of B-cells.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of an aptamer (see, e.g., Ellington etal., Nature. 346, 818-22, 1990; and Tuerk et al., Science. 249, 505-10,1990, incorporated by reference). Examples of aptamers included nucleicacid aptamers (e.g., DNA aptamers, RNA aptamers) and peptide aptamers.Nucleic acid aptamers refer generally to nucleic acid species that havebeen engineered through repeated rounds of in vitro selection orequivalent method, such as SELEX (systematic evolution of ligands byexponential enrichment), to bind to various molecular targets such assmall molecules, proteins, nucleic acids, and even cells, tissues andorganisms. See, e.g., U.S. Pat. Nos. 6,376,190; and 6,387,620,incorporated by reference.

Peptide aptamers typically include a variable peptide loop attached atboth ends to a protein scaffold, a double structural constraint thattypically increases the binding affinity of the peptide aptamer tolevels comparable to that of an antibody's (e.g., in the nanomolarrange). In certain embodiments, the variable loop length may be composedof about 10-20 amino acids (including all integers in between), and thescaffold may include any protein that has good solubility and compacityproperties. Certain exemplary embodiments utilize the bacterial proteinThioredoxin-A as a scaffold protein, the variable loop being insertedwithin the reducing active site (-Cys-Gly-Pro-Cys-loop in the wildprotein), with the two cysteines lateral chains being able to form adisulfide bridge. Methods for identifying peptide aptamers aredescribed, for example, in U.S. Application No. 2003/0108532,incorporated by reference. Peptide aptamer selection can be performedusing different systems known in the art, including the yeast two-hybridsystem.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of an avimer. Avimers refer tomultimeric binding proteins or peptides engineered using in vitro exonshuffling and phage display. Multiple binding domains are linked,resulting in greater affinity and specificity compared to single epitopeimmunoglobulin domains. See, e.g., Silverman et al., NatureBiotechnology. 23:1556-1561, 2005; U.S. Pat. No. 7,166,697; and U.S.Application Nos. 2004/0175756, 2005/0048512, 2005/0053973, 2005/0089932and 2005/0221384, incorporated by reference.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of an adnectin. Adnectins refer to aclass of targeted biologics derived from human fibronectin, an abundantextracellular protein that naturally binds to other proteins. See, e.g.,U.S. Application Nos. 2007/0082365; 2008/0139791; and 2008/0220049,incorporated by reference. Adnectins typically consists of a naturalfibronectin backbone, as well as the multiple targeting domains of aspecific portion of human fibronectin. The targeting domains can beengineered to enable an adnectin to specifically recognize an NRP2polypeptide or an epitope thereof.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of an anticalin. Anticalins refer to aclass of antibody mimetics that are typically synthesized from humanlipocalins, a family of binding proteins with a hypervariable loopregion supported by a structurally rigid framework. See, e.g., U.S.Application No. 2006/0058510. Anticalins typically have a size of about20 kDa. Anticalins can be characterized by a barrel structure formed byeight antiparallel β-strands (a stable β-barrel scaffold) that arepairwise connected by four peptide loops and an attached α-helix. Incertain aspects, conformational deviations to achieve specific bindingare made in the hypervariable loop region(s). See, e.g., Skerra, FEBS J.275:2677-83, 2008, incorporated by reference.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein are in the form of a designed ankyrin repeat protein(DARPin). DARPins include a class of non-immunoglobulin proteins thatcan offer advantages over antibodies for target binding in drugdiscovery and drug development. Among other uses, DARPins are ideallysuited for in vivo imaging or delivery of toxins or other therapeuticpayloads because of their favorable molecular properties, includingsmall size and high stability. The low-cost production in bacteria andthe rapid generation of many target-specific DARPins make the DARPinapproach useful for drug discovery. Additionally, DARPins can be easilygenerated in multispecific formats, offering the potential to target aneffector DARPin to a specific organ or to target multiple receptors withone molecule composed of several DARPins. See, e.g., Stumpp et al., CurrOpin Drug Discov Devel. 10:153-159, 2007; U.S. Application No.2009/0082274; and PCT/EP2001/10454, incorporated by reference.

Also included are heavy chain dimers, such as antibodies from camelidsand sharks. Camelid and shark antibodies comprise a homodimeric pair oftwo chains of V-like and C-like domains (neither has a light chain).Since the VH region of a heavy chain dimer IgG in a camelid does nothave to make hydrophobic interactions with a light chain, the region inthe heavy chain that normally contacts a light chain is changed tohydrophilic amino acid residues in a camelid. VH domains of heavy-chaindimer IgGs are called VHH domains. Shark Ig-NARs comprise a homodimer ofone variable domain (termed a V-NAR domain) and five C-like constantdomains (C-NAR domains).

In camelids, the diversity of antibody repertoire is determined by thecomplementary determining regions (CDR) 1, 2, and 3 in the VH or VHHregions. The CDR3 in the camel VHH region is characterized by itsrelatively long length averaging 16 amino acids (Muyldermans et al.,1994, Protein Engineering 7(9): 1129). This is in contrast to CDR3regions of antibodies of many other species. For example, the CDR3 ofmouse VH has an average of 9 amino acids. Libraries of camelid-derivedantibody variable regions, which maintain the in vivo diversity of thevariable regions of a camelid, can be made by, for example, the methodsdisclosed in U.S. Patent Application Ser. No. 20050037421, publishedFeb. 17, 2005

In certain embodiments, the antibodies or antigen-binding fragmentsthereof are humanized. These embodiments refer to a chimeric molecule,generally prepared using recombinant techniques, having anantigen-binding site derived from an immunoglobulin from a non-humanspecies and the remaining immunoglobulin structure of the molecule basedupon the structure and/or sequence of a human immunoglobulin. Theantigen-binding site may comprise either complete variable domains fusedonto constant domains or only the CDRs grafted onto appropriateframework regions in the variable domains. Epitope binding sites may bewild type or modified by one or more amino acid substitutions. Thiseliminates the constant region as an immunogen in human individuals, butthe possibility of an immune response to the foreign variable regionremains (LoBuglio et al., PNAS USA 86:4220-4224, 1989; Queen et al.,PNAS USA. 86:10029-10033, 1988; Riechmann et al., Nature. 332:323-327,1988). Illustrative methods for humanization of antibodies include themethods described in U.S. Pat. No. 7,462,697.

Another approach focuses not only on providing human-derived constantregions, but modifying the variable regions as well so as to reshapethem as closely as possible to human form. It is known that the variableregions of both heavy and light chains contain threecomplementarity-determining regions (CDRs) which vary in response to theepitopes in question and determine binding capability, flanked by fourframework regions (FRs) which are relatively conserved in a givenspecies and which putatively provide a scaffolding for the CDRs. Whennonhuman antibodies are prepared with respect to a particular epitope,the variable regions can be “reshaped” or “humanized” by grafting CDRsderived from nonhuman antibody on the FRs present in the human antibodyto be modified. Application of this approach to various antibodies hasbeen reported by Sato et al., Cancer Res. 53:851-856, 1993; Riechmann etal., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536,1988; Kettleborough et al., Protein Engineering. 4:773-3783, 1991; Maedaet al., Human Antibodies Hybridoma 2:124-134, 1991; Gorman et al., PNASUSA. 88:4181-4185, 1991; Tempest et al., Bio/Technology 9:266-271, 1991;Co et al., PNAS USA. 88:2869-2873, 1991; Carter et al., PNAS USA.89:4285-4289, 1992; and Co et al., J Immunol. 148:1149-1154, 1992. Insome embodiments, humanized antibodies preserve all CDR sequences (forexample, a humanized mouse antibody which contains all six CDRs from themouse antibodies). In other embodiments, humanized antibodies have oneor more CDRs (one, two, three, four, five, six) which are altered withrespect to the original antibody, which are also termed one or more CDRs“derived from” one or more CDRs from the original antibody.

In certain embodiments, the antibodies are “chimeric” antibodies. Inthis regard, a chimeric antibody is comprised of an antigen-bindingfragment of an antibody operably linked or otherwise fused to aheterologous Fc portion of a different antibody. In certain embodiments,the Fc domain or heterologous Fc domain is of human origin. In certainembodiments, the Fc domain or heterologous Fc domain is of mouse origin.In other embodiments, the heterologous Fc domain may be from a differentIg class from the parent antibody, including IgA (including subclassesIgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3,and IgG4), and IgM. In further embodiments, the heterologous Fc domainmay be comprised of CH2 and CH3 domains from one or more of thedifferent Ig classes. As noted above with regard to humanizedantibodies, the antigen-binding fragment of a chimeric antibody maycomprise only one or more of the CDRs of the antibodies described herein(e.g., 1, 2, 3, 4, 5, or 6 CDRs of the antibodies described herein), ormay comprise an entire variable domain (VL, VH or both).

As used herein, a subject “at risk” of developing a disease, or adversereaction may or may not have detectable disease, or symptoms of disease,and may or may not have displayed detectable disease or symptoms ofdisease prior to the treatment methods described herein. “At risk”denotes that a subject has one or more risk factors, which aremeasurable parameters that correlate with development of a disease, asdescribed herein and known in the art. A subject having one or more ofthese risk factors has a higher probability of developing disease, or anadverse reaction than a subject without one or more of these riskfactor(s).

“Biocompatible” refers to materials or compounds which are generally notinjurious to biological functions of a cell or subject and which willnot result in any degree of unacceptable toxicity, including allergenicand disease states.

The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, and ionicand/or hydrogen-bond interactions, including interactions such as saltbridges and water bridges.

The term “chemoresistance” refers to the change in therapeuticsensitivity of a cancer cell population over time following exposure tochemotherapy, including resistance to at least one of a cancerimmunotherapy agent, a chemotherapeutic agent, a hormonal therapeuticagent, and/or a kinase inhibitor. Eventually, chemoresistance leads tothe relapse and/or metastasis, of the cancer, and challenges theimprovement of clinical outcome for the cancer patients. It remains themain obstacle to long term successful cancer therapy. For example,approximately 30 percent of women diagnosed with early-stage breastcancer ultimately develop resistance and eventually progress tometastatic breast cancer. The molecular mechanisms of chemoresistanceinclude the induction of transporter pumps, oncogenes, tumor suppressorgenes, mitochondrial alteration, DNA repair, autophagy,epithelial-mesenchymal transition (EMT), cancer stemness, and exosomeproduction. These processes may operate via distinct mechanisms, aloneor in combination with each other, but ultimately coordinate to preventcell death in response to a specific targeted chemotherapeutic agent.For example, such processes provide alternative pro-growth signalsand/or eliminate or otherwise reduce apoptotic pathways. Accordingly,agents that reduce chemoresistance could find utility in the treatmentor reduction of chemoresistant cancers.

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does notdirectly contribute to the code for the polypeptide product of a gene.

Throughout this disclosure, unless the context requires otherwise, thewords “comprise,” “comprises,” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of.” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they materiallyaffect the activity or action of the listed elements.

The term “effector function”, or “ADCC effector function” in the contextof antibodies refers to the ability of that antibody to engage withother arms of the immune system, including for example, the activationof the classical complement pathway, or through engagement of Fcreceptors. Complement dependent pathways are primarily driven by theinteraction of C1q with the C1 complex with clustered antibody Fcdomains. Antibody dependent cellular cytotoxicity (ADCC), is primarilydriven by the interaction of Fc receptors (FcRs) on the surface ofeffector cells (natural killer cells, macrophages, monocytes andeosinophils) which bind to the Fc region of an IgG which itself is boundto a target cell. Fc receptors (FcRs) are key immune regulatoryreceptors connecting the antibody mediated (humoral) immune response tocellular effector functions. Receptors for all classes ofimmunoglobulins have been identified, including FcγR (IgG), FCεRI (IgE),FcαRI (IgA), FcμR (IgM) and FcδR (IgD). There are at least three classesof receptors for human IgG found on leukocytes: CD64 (FcγRI), CD32(FcγRIIa, FcγRIIb and FcγRIIc) and CD16 (FcγIIIa and FcγRIIIb). FcγRI isclassed as a high affinity receptor (nanomolar range KD) while FcγRIIand FcγRII are low to intermediate affinity (micromolar range KD). UponFc binding a signaling pathway is triggered which results in thesecretion of various substances, such as lytic enzymes, perforin,granzymes and tumour necrosis factor, which mediate in the destructionof the target cell. The level of ADCC effector function various forhuman IgG subtypes. Although this is dependent on the allotype andspecific FcvR, in simple terms ADCC effector function is “high” forhuman IgG1 and IgG3, and “low” for IgG2 and IgG4.

The term “endotoxin free” or “substantially endotoxin free” relatesgenerally to compositions, solvents, and/or vessels that contain at mosttrace amounts (e.g., amounts having no clinically adverse physiologicaleffects to a subject) of endotoxin, and preferably undetectable amountsof endotoxin. Endotoxins are toxins associated with certainmicro-organisms, such as bacteria, typically gram-negative bacteria,although endotoxins may be found in gram-positive bacteria, such asListeria monocytogenes. The most prevalent endotoxins arelipopolysaccharides (LPS) or lipo-oligo-saccharides (LOS) found in theouter membrane of various Gram-negative bacteria, and which represent acentral pathogenic feature in the ability of these bacteria to causedisease. Small amounts of endotoxin in humans may produce fever, alowering of the blood pressure, and activation of inflammation andcoagulation, among other adverse physiological effects.

Therefore, in pharmaceutical production, it is often desirable to removemost or all traces of endotoxin from drug products and/or drugcontainers, because even small amounts may cause adverse effects inhumans. A depyrogenation oven may be used for this purpose, astemperatures in excess of 300° C. are typically required to break downmost endotoxins. For instance, based on primary packaging material suchas syringes or vials, the combination of a glass temperature of 250° C.and a holding time of 30 minutes is often sufficient to achieve a 3 logreduction in endotoxin levels. Other methods of removing endotoxins arecontemplated, including, for example, chromatography and filtrationmethods, as described herein and known in the art.

Endotoxins can be detected using routine techniques known in the art.For example, the Limulus Amoebocyte Lysate assay, which utilizes bloodfrom the horseshoe crab, is a very sensitive assay for detectingpresence of endotoxin. In this test, very low levels of LPS can causedetectable coagulation of the limulus lysate due a powerful enzymaticcascade that amplifies this reaction. Endotoxins can also be quantitatedby enzyme-linked immunosorbent assay (ELISA). To be substantiallyendotoxin free, endotoxin levels may be less than about 0.001, 0.005,0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2,2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU/mg of active compound. Typically, 1ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. An epitope includes a region of an antigen that is bound by anantibody. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl or sulfonyl, and may in certainembodiments have specific three-dimensional structural characteristics,and/or specific charge characteristics. Epitopes can be contiguous ornon-contiguous in relation to the primary structure of the antigen, forexample, an NRP2 polypeptide. In particular embodiments, an epitopecomprises, consists, or consists essentially of about, at least about,or no more than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 contiguous amino acids (i.e., a linear epitope) ornon-contiguous amino acids (i.e., conformational epitope) of a referencesequence (see, e.g., Table N1) or target molecule described herein.

An “epitope” includes that portion of an antigen or other macromoleculecapable of forming a binding interaction that interacts with thevariable region binding pocket of a binding protein. Such bindinginteraction can be manifested as an intermolecular contact with one ormore amino acid residues of a CDR. Antigen binding can involve a CDR3 ora CDR3 pair. An epitope can be a linear peptide sequence (i.e.,“continuous”) or can be composed of noncontiguous amino acid sequences(i.e., “conformational” or “discontinuous”). A binding protein canrecognize one or more amino acid sequences; therefore an epitope candefine more than one distinct amino acid sequence. Epitopes recognizedby binding protein can be determined by peptide mapping and sequenceanalysis techniques well known to one of skill in the art. A “crypticepitope” or a “cryptic binding site” is an epitope or binding site of aprotein sequence that is not exposed or substantially protected fromrecognition within an unmodified polypeptide, but is capable of beingrecognized by a binding protein of a denatured or proteolyzedpolypeptide. Amino acid sequences that are not exposed, or are onlypartially exposed, in the unmodified polypeptide structure are potentialcryptic epitopes. If an epitope is not exposed, or only partiallyexposed, then it is likely that it is buried within the interior of thepolypeptide. Candidate cryptic epitopes can be identified, for example,by examining the three-dimensional structure of an unmodifiedpolypeptide.

The term “half maximal effective concentration” or “EC₅₀” refers to theconcentration of an agent (e.g., antibody) as described herein at whichit induces a response halfway between the baseline and maximum aftersome specified exposure time; the EC₅₀ of a graded dose response curvetherefore represents the concentration of a compound at which 50% of itsmaximal effect is observed. EC50 also represents the plasmaconcentration required for obtaining 50% of a maximum effect in vivo.Similarly, the “EC₉₀” refers to the concentration of an agent orcomposition at which 90% of its maximal effect is observed. The “EC₉₀”can be calculated from the “EC50” and the Hill slope, or it can bedetermined from the data directly, using routine knowledge in the art.In some embodiments, the EC50 of an agent (e.g., antibody) is less thanabout 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,40, 50, 60, 70, 80, 90, 100, 200 or 500 nM. In some embodiments, anagent will have an EC₅₀ value of about 1 nM or less.

“Immune response” means any immunological response originating fromimmune system, including responses from the cellular and humeral, innateand adaptive immune systems. Exemplary cellular immune cells include forexample, lymphocytes, macrophages, T cells, B cells, NK cells,neutrophils, eosinophils, dendritic cells, mast cells, monocytes, andall subsets thereof. Cellular responses include for example, effectorfunction, cytokine release, phagocytosis, efferocytosis, translocation,trafficking, proliferation, differentiation, activation, repression,cell-cell interactions, apoptosis, etc. Humeral responses include forexample IgG, IgM, IgA, IgE, responses and their corresponding effectorfunctions.

The “half-life” of an agent such as an antibody can refer to the time ittakes for the agent to lose half of its pharmacologic, physiologic, orother activity, relative to such activity at the time of administrationinto the serum or tissue of an organism, or relative to any otherdefined time-point. “Half-life” can also refer to the time it takes forthe amount or concentration of an agent to be reduced by half of astarting amount administered into the serum or tissue of an organism,relative to such amount or concentration at the time of administrationinto the serum or tissue of an organism, or relative to any otherdefined time-point. The half-life can be measured in serum and/or anyone or more selected tissues.

The terms “modulating” and “altering” include “increasing,” “enhancing”or “stimulating,” as well as “decreasing” or “reducing,” typically in astatistically significant or a physiologically significant amount ordegree relative to a control. An “increased,” “stimulated” or “enhanced”amount is typically a “statistically significant” amount, and mayinclude an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more times (e.g., 500, 1000times) (including all integers and ranges in between e.g., 1.5, 1.6,1.7. 1.8, etc.) the amount produced by no composition (e.g., the absenceof agent) or a control composition. A “decreased” or “reduced” amount istypically a “statistically significant” amount, and may include a 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% decrease (including all integers and rangesin between) in the amount produced by no composition (e.g., the absenceof an agent) or a control composition. Examples of comparisons and“statistically significant” amounts are described herein.

The term “migratory cells” refers to cells that are capable of movementfrom one place to another in response to a stimulus. Exemplary migratorycells include immune cells such as monocytes, Natural Killer (NK) cells,dendritic cells (immature or mature), subsets of dendritic cellsincluding myeloid, plasmacytoid (also called lymphoid) and Langerhanscells, macrophages such as histiocytes, tissue resident macrophages suchas Kupffer's cells, microglia cells in the CNS, alveolar macrophages,and peritoneal macrophages, macrophage subtypes such as M0, M1, Mox,M2a, M2b, and M2c macrophages, neutrophils, eosinophils, mast cells,basophils, B cells including plasma B cells, memory B cells, B-1 cells,and B-2 cells, CD45RO (naive T) cells, CD45RA (memory T) cells, CD4Helper T Cells including Th1, Th2, and Tr1/Th3 cells, CD8 Cytotoxic TCells, Regulatory T Cells, Gamma Delta T Cells, and thymocytes.Additional examples of migratory cells include fibroblasts, fibrocytes,tumor cells, and stem cells. The term “cell migration” refers to themovement of migratory cells, and the term “modulation of cell migration”refers to the modulation of the movement of any such migratory cells.

The terms “polypeptide,” “protein” and “peptide” are usedinterchangeably and mean a polymer of amino acids not limited to anyparticular length. The term “enzyme” includes polypeptide or proteincatalysts. The terms include modifications such as myristoylation,sulfation, glycosylation, phosphorylation and addition or deletion ofsignal sequences. The terms “polypeptide” or “protein” means one or morechains of amino acids, wherein each chain comprises amino acidscovalently linked by peptide bonds, and wherein said polypeptide orprotein can comprise a plurality of chains non-covalently and/orcovalently linked together by peptide bonds, having the sequence ofnative proteins, that is, proteins produced by naturally-occurring andspecifically non-recombinant cells, or genetically-engineered orrecombinant cells, and comprise molecules having the amino acid sequenceof the native protein, or molecules having deletions from, additions to,and/or substitutions of one or more amino acids of the native sequence.In certain embodiments, the polypeptide is a “recombinant” polypeptide,produced by recombinant cell that comprises one or more recombinant DNAmolecules, which are typically made of heterologous polynucleotidesequences or combinations of polynucleotide sequences that would nototherwise be found in the cell.

The term “polynucleotide” and “nucleic acid” includes mRNA, RNA, cRNA,cDNA, and DNA. The term typically refers to polymeric form ofnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA. The terms“isolated DNA” and “isolated polynucleotide” and “isolated nucleic acid”refer to a molecule that has been isolated free of total genomic DNA ofa particular species. Therefore, an isolated DNA segment encoding apolypeptide refers to a DNA segment that contains one or more codingsequences yet is substantially isolated away from, or purified freefrom, total genomic DNA of the species from which the DNA segment isobtained. Also included are non-coding polynucleotides (e.g., primers,probes, oligonucleotides), which do not encode a polypeptide. Alsoincluded are recombinant vectors, including, for example, expressionvectors, viral vectors, plasmids, cosmids, phagemids, phage, viruses,and the like.

Additional coding or non-coding sequences may, but need not, be presentwithin a polynucleotide described herein, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials. Hence,a polynucleotide or expressible polynucleotides, regardless of thelength of the coding sequence itself, may be combined with othersequences, for example, expression control sequences.

“Expression control sequences” include regulatory sequences of nucleicacids, or the corresponding amino acids, such as promoters, leaders,enhancers, introns, recognition motifs for RNA, or DNA binding proteins,polyadenylation signals, terminators, internal ribosome entry sites(IRES), secretion signals, subcellular localization signals, and thelike, which have the ability to affect the transcription or translation,or subcellular, or cellular location of a coding sequence in a hostcell. Exemplary expression control sequences are described in Goeddel;Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990).

A “promoter” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. As used herein, the promoter sequence isbounded at its 3′ terminus by the transcription initiation site andextends upstream (5′ direction) to include the minimum number of basesor elements necessary to initiate transcription at levels detectableabove background. A transcription initiation site (conveniently definedby mapping with nuclease S1) can be found within a promoter sequence, aswell as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. Eukaryotic promoters can often, but notalways, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoterscontain Shine-Dalgarno sequences in addition to the −10 and −35consensus sequences.

A large number of promoters, including constitutive, inducible andrepressible promoters, from a variety of different sources are wellknown in the art. Representative sources include for example, viral,mammalian, insect, plant, yeast, and bacterial cell types), and suitablepromoters from these sources are readily available, or can be madesynthetically, based on sequences publicly available on line or, forexample, from depositories such as the ATCC as well as other commercialor individual sources. Promoters can be unidirectional (i.e., initiatetranscription in one direction) or bi-directional (i.e., initiatetranscription in either a 3′ or 5′ direction). Non-limiting examples ofpromoters include, for example, the T7 bacterial expression system, pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter,the SV40 promoter, the RSV promoter. Inducible promoters include the Tetsystem, (U.S. Pat. Nos. 5,464,758 and 5,814,618), the Ecdysone induciblesystem (No et al., Proc. Natl. Acad. Sci. (1996) 93 (8): 3346-3351; theT-REx™ system (Invitrogen Carlsbad, Calif.), LacSwitch® (Stratagene,(San Diego, Calif.) and the Cre-ERT tamoxifen inducible recombinasesystem (Indra et al. Nuc. Acid. Res. (1999) 27 (22): 4324-4327; Nuc.Acid. Res. (2000) 28 (23): e99; U.S. Pat. No. 7,112,715; and Kramer &Fussenegger, Methods Mol. Biol. (2005) 308: 123-144) or any promoterknown in the art suitable for expression in the desired cells.

An “expressible polynucleotide” includes a cDNA, RNA, mRNA or otherpolynucleotide that comprises at least one coding sequence andoptionally at least one expression control sequence, for example, atranscriptional and/or translational regulatory element, and which canexpress an encoded polypeptide upon introduction into a cell, forexample, a cell in a subject.

Various viral vectors that can be utilized to deliver an expressiblepolynucleotide include adenoviral vectors, herpes virus vectors,vaccinia virus vectors, adeno-associated virus (AAV) vectors, andretroviral vectors. In some instances, the retroviral vector is aderivative of a murine or avian retrovirus, or is a lentiviral vector.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A number ofadditional retroviral vectors can incorporate multiple genes. All ofthese vectors can transfer or incorporate a gene for a selectable markerso that transduced cells can be identified and generated. By inserting apolypeptide sequence of interest into the viral vector, along withanother gene that encodes the ligand for a receptor on a specific targetcell, for example, the vector may be made target specific. Retroviralvectors can be made target specific by inserting, for example, apolynucleotide encoding a protein. Illustrative targeting may beaccomplished by using an antibody to target the retroviral vector. Thoseof skill in the art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome to allow target specific delivery of theretroviral vector.

In particular embodiments, the expressible polynucleotide is a modifiedRNA or modified mRNA polynucleotide, for example, a non-naturallyoccurring RNA analog. In certain embodiments, the modified RNA or mRNApolypeptide comprises one or more modified or non-natural bases, forexample, a nucleotide base other than adenine (A), guanine (G), cytosine(C), thymine (T), and/or uracil (U). In some embodiments, the modifiedmRNA comprises one or more modified or non-natural internucleotidelinkages. Expressible RNA polynucleotides for delivering an encodedtherapeutic polypeptide are described, for example, in Kormann et al.,Nat Biotechnol. 29:154-7, 2011; and U.S. Application Nos. 2015/0111248;2014/0243399; 2014/0147454; and 2013/0245104, which are incorporated byreference in their entireties.

The term “isolated” polypeptide or protein referred to herein means thata subject protein (1) is free of at least some other proteins with whichit would typically be found in nature, (2) is essentially free of otherproteins from the same source, e.g., from the same species, (3) isexpressed by a cell from a different species, (4) has been separatedfrom at least about 50 percent of polynucleotides, lipids,carbohydrates, or other materials with which it is associated in nature,(5) is not associated (by covalent or non-covalent interaction) withportions of a protein with which the “isolated protein” is associated innature, (6) is operably associated (by covalent or non-covalentinteraction) with a polypeptide with which it is not associated innature, or (7) does not occur in nature. Such an isolated protein can beencoded by genomic DNA, cDNA, mRNA or other RNA, of may be of syntheticorigin, or any combination thereof. In certain embodiments, the isolatedprotein is substantially free from proteins or polypeptides or othercontaminants that are found in its natural environment that wouldinterfere with its use (therapeutic, diagnostic, prophylactic, researchor otherwise).

In certain embodiments, the “purity” of any given agent (e.g.,polypeptide such as an antibody) in a composition may be defined. Forinstance, certain compositions may comprise an agent such as apolypeptide agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% pure on a protein basis or aweight-weight basis, including all decimals and ranges in between, asmeasured, for example and by no means limiting, by high performanceliquid chromatography (HPLC), a well-known form of column chromatographyused frequently in biochemistry and analytical chemistry to separate,identify, and quantify compounds.

A “lipid nanoparticle” or “solid lipid nanoparticle” refers to one ormore spherical nanoparticles with an average diameter of between about10 to about 1000 nanometers, and which comprise a solid lipid corematrix that can solubilize lipophilic molecules. The lipid core isstabilized by surfactants (e.g., emulsifiers), and can comprise one ormore of triglycerides (e.g., tristearin), diglycerides (e.g., glycerolbahenate), monoglycerides (e.g., glycerol monostearate), fatty acids(e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g.,cetyl palmitate), including combinations thereof. Lipid nanoparticlesare described, for example, in Petrilli et al., Curr Pharm Biotechnol.15:847-55, 2014; and U.S. Pat. Nos. 6,217,912; 6,881,421; 7,402,573;7,404,969; 7,550,441; 7,727,969; 8,003,621; 8,691,750; 8,871,509;9,017,726; 9,173,853; 9,220,779; 9,227,917; and 9,278,130, which areincorporated by reference in their entireties. Certain compositionsdescribed herein are formulated with one or more lipid nanoparticles.

The terms or “Neuropilin 2-associated disease” or “NRP2-associateddisease” refer to diseases and conditions in which NRP2 activity,expression, and/or spatial distribution plays a role in thepathophysiology of that disease or condition. In some instances, NRP2associated diseases are modulated by the anti-NRP2 antibodies of thepresent disclosure by altering the interaction of NRP2 with at least oneNRP2 ligand to impact NRP2 activity, signaling, expression, and/orspatial distribution. Exemplary NRP2-associated diseases and conditionsinclude without limitation, cancer and diseases or pathologiesassociated with cancer including cancer cell growth, cancer initiation,cancer migration, cancer cell adhesion, invasion, chemoresistance, andmetastasis. Also included are diseases associated with inflammation andautoimmunity, and related inflammatory diseases, including diseaseassociated with inappropriate immune cell activation or migration suchas graft versus host disease (GVHD). Additional examples includediseases associated with lymphatic development, lymphangiogenesis, andlymphatic damage, including edema, lymphedema, secondary lymphedema,inappropriate fat absorption and deposition, excess fat deposition, andvascular permeability. Also included are diseases associated withinfections including latent infections, and diseases associated withallergic disorders/diseases and allergic responses, including chronicobstructive pulmonary disorder (COPD), neutrophilic asthma,antineutrophil cytoplasmic antibody (ANCA)-associated systemicvasculitis, systemic lupus erythematosus, rheumatoid arthritis,inflammasome-related disease(s), and skin-related neutrophil-mediateddisease(s) such as pyoderma gangrenosum. Additional examples includediseases associated with granulomatous inflammatory diseases includingsarcoidosis and granulomas, and fibrotic diseases includingendometriosis, fibrosis, endothelial to mesenchymal transition (EMT),and wound healing, among others. Also included are diseases associatedwith inappropriate smooth muscle contractility, smooth musclecompensation and decompensation, vascular smooth muscle cell migrationand/or adhesion, and diseases associated with inappropriate autophagy,phagocytosis, and efferocytosis. Also included are diseases associatedwith inappropriate migratory cell movement, as described herein.Additional examples include neuronal diseases, including diseasesassociated with peripheral nervous system remodeling and painperception. Also included are diseases associated with bone developmentand/or bone remodeling. Typically, the term “inappropriate” refers to anactivity or characteristic that associates with or causes a pathology ordisease state.

The term “reference sequence” refers generally to a nucleic acid codingsequence, or amino acid sequence, to which another sequence is beingcompared. All polypeptide and polynucleotide sequences described hereinare included as references sequences, including those described by nameand those described in the Tables and the Sequence Listing.

Certain embodiments include biologically active “variants” and“fragments” of the polypeptides (e.g., antibodies) described herein, andthe polynucleotides that encode the same. “Variants” contain one or moresubstitutions, additions, deletions, and/or insertions relative to areference polypeptide or polynucleotide (see, e.g., the Tables and theSequence Listing). A variant polypeptide or polynucleotide comprises anamino acid or nucleotide sequence with at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity or similarity or homology to a referencesequence, as described herein, and substantially retains the activity ofthat reference sequence. Also included are sequences that consist of ordiffer from a reference sequences by the addition, deletion, insertion,or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150 or more amino acids or nucleotides and which substantially retainthe activity of that reference sequence. In certain embodiments, theadditions or deletions include C-terminal and/or N-terminal additionsand/or deletions.

The terms “sequence identity” or, for example, comprising a “sequence50% identical to,” as used herein, refer to the extent that sequencesare identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. Optimal alignment of sequences for aligning a comparisonwindow may be conducted by computerized implementations of algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, 575 Science Drive Madison,Wis., USA) or by inspection and the best alignment (i.e., resulting inthe highest percentage homology over the comparison window) generated byany of the various methods selected. Reference also may be made to theBLAST family of programs as for example disclosed by Altschul et al.,Nucl. Acids Res. 25:3389, 1997.

The term “solubility” refers to the property of an agent (e.g.,antibody) provided herein to dissolve in a liquid solvent and form ahomogeneous solution. Solubility is typically expressed as aconcentration, either by mass of solute per unit volume of solvent (g ofsolute per kg of solvent, g per dL (100 mL), mg/ml, etc.), molarity,molality, mole fraction or other similar descriptions of concentration.The maximum equilibrium amount of solute that can dissolve per amount ofsolvent is the solubility of that solute in that solvent under thespecified conditions, including temperature, pressure, pH, and thenature of the solvent. In certain embodiments, solubility is measured atphysiological pH, or other pH, for example, at pH 5.0, pH 6.0, pH 7.0,pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5-8). In certainembodiments, solubility is measured in water or a physiological buffersuch as PBS or NaCl (with or without NaPO₄). In specific embodiments,solubility is measured at relatively lower pH (e.g., pH 6.0) andrelatively higher salt (e.g., 500 mM NaCl and 10 mM NaPO₄). In certainembodiments, solubility is measured in a biological fluid (solvent) suchas blood or serum. In certain embodiments, the temperature can be aboutroom temperature (e.g., about 20, 21, 22, 23, 24, 25° C.) or about bodytemperature (37° C.). In certain embodiments, an agent has a solubilityof at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,40, 50, 60, 70, 80, 90 or 100 mg/ml at room temperature or at 37° C.

A “subject” or a “subject in need thereof” or a “patient” or a “patientin need thereof” includes a mammalian subject such as a human subject.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

By “statistically significant,” it is meant that the result was unlikelyto have occurred by chance. Statistical significance can be determinedby any method known in the art. Commonly used measures of significanceinclude the p-value, which is the frequency or probability with whichthe observed event would occur, if the null hypothesis were true. If theobtained p-value is smaller than the significance level, then the nullhypothesis is rejected. In simple cases, the significance level isdefined at a p-value of 0.05 or less.

“Therapeutic response” refers to improvement of symptoms (whether or notsustained) based on administration of one or more therapeutic agents.

As used herein, the terms “therapeutically effective amount”,“therapeutic dose,” “prophylactically effective amount,” or“diagnostically effective amount” is the amount of an agent (e.g.,anti-NRP2 antibody, immunotherapy agent) needed to elicit the desiredbiological response following administration.

As used herein, “treatment” of a subject (e.g., a mammal, such as ahuman) or a cell is any type of intervention used in an attempt to alterthe natural course of the individual or cell. Treatment includes, but isnot limited to, administration of a pharmaceutical composition, and maybe performed either prophylactically or subsequent to the initiation ofa pathologic event or contact with an etiologic agent. Also included are“prophylactic” treatments, which can be directed to reducing the rate ofprogression of the disease or condition being treated, delaying theonset of that disease or condition, or reducing the severity of itsonset. “Treatment” or “prophylaxis” does not necessarily indicatecomplete eradication, cure, or prevention of the disease or condition,or associated symptoms thereof.

The term “wild-type” refers to a gene or gene product (e.g., apolypeptide) that is most frequently observed in a population and isthus arbitrarily designed the “normal” or “wild-type” form of the gene.

Each embodiment in this specification is to be applied to every otherembodiment unless expressly stated otherwise.

Anti-NRP2 Antibodies

Certain embodiments include antibodies, and antigen-binding fragmentsthereof, which specifically bind to a human neuropilin 2 (NRP2)polypeptide. In some embodiments, the at least one antibody orantigen-binding fragment thereof modulates (e.g., interferes with)binding of the human NRP2 polypeptide to at least one NRP2 ligand, suchas a human histidyl-tRNA synthetase (HRS) polypeptide or other NRP2ligand.

In certain embodiments, an antibody or antigen-binding fragment thereofis characterized by or comprises a heavy chain variable region (V_(H))sequence that comprises complementary determining region V_(H)CDR1,V_(H)CDR2, and V_(H)CDR3 sequences, and a light chain variable region(V_(L)) sequence that comprises complementary determining regionV_(L)CDR1, V_(L)CDR2, and V_(L)CDR3 sequences. Exemplary V_(H),V_(H)CDR1, V_(H)CDR2, V_(H)CDR3, V_(L), V_(L)CDR1, V_(L)CDR2, andV_(L)CDR3 sequences are provided in Table A1, Table A2, and Table A3below.

TABLE A1 Exemplary CDR Sequences SEQ ID Description Sequence NO: aNRP2-1v3 V_(H)CDR1 GYTFTSYWMH 1 V_(H)CDR2 AIYPGNSDTSYNQQFKG 2 V_(H)CDR3RGGGYFDY 3 V_(L)CDR1 KASQNVGAAVA 4 V_(L)CDR2 SASNRYT 5 V_(L)CDR3QQYSSYPLLT 6 aNRP2-2v4 V_(H)CDR1 GYTFTSYWMH 7 V_(H)CDR2VIHPNSASTFYNERFKT 8 V_(H)CDR3 PGTVRRSDY 9 V_(L)CDR1 RSSQNIVHSTGNTYLE 10V_(L)CDR2 KVSNRFS 11 V_(L)CDR3 FQGSHVPWT 12 aNRP2-10v5 V_(H)CDR1GFNIKDYYIH 13 V_(H)CDR2 RIDVEDDETKYAPKFQG 14 V_(H)CDR3 PIYGSREAWFAY 15V_(L)CDR1 TASSSVSSSYLH 16 V_(L)CDR2 RTSNLAS 17 V_(L)CDR3 HQYYRSPPT 18aNRP2-10v10 V_(H)CDR1 GFNIKDYYVH 19 V_(H)CDR2 RIDVEDDETKYAPKFQG 20V_(H)CDR3 PIYGAREAWFAY 21 V_(L)CDR1 TASSSVSSSYLH 22 V_(L)CDR2 RTSNLAS 23V_(L)CDR3 HQYYRSPPT 24 aNRP2-10v13 V_(H)CDR1 GFNIKDYYVH 57 V_(H)CDR2RIDVHDDETKYAPKFQG 58 V_(H)CDR3 PIYGAREAWFAY 59 V_(L)CDR1 TASSSVSSSYLH 60V_(L)CDR2 RTSNLAS 61 V_(L)CDR3 HQYYRSPPT 62 aNRP2-11v7 V_(H)CDR1GYTFTSFGIS 25 V_(H)CDR2 EIYPRSGNTYYNENFKG 26 V_(H)CDR3 SSGYYGSTPFPY 27V_(L)CDR1 RASQDISNYLN 28 V_(L)CDR2 YTSRLHS 29 V_(L)CDR3 QQGNTLPWT 30aNRP2-14v9 V_(H)CDR1 GFSLTSYGVH 31 V_(H)CDR2 LIWSGGSTDYSPAFIS 32V_(H)CDR3 NSYSSGYYAMDY 33 V_(L)CDR1 KASQNVGHAVA 34 V_(L)CDR2 SASNRYT 35V_(L)CDR3 QQYSRYPPYT 36 aNRP2-14v10 V_(H)CDR1 GFSLTSYGVH 37 V_(H)CDR2LIWSGGSTDYSPAFIS 38 V_(H)CDR3 NSYSSGYYAMDY 39 V_(L)CDR1 KASQNVGTAVA 40V_(L)CDR2 SASNRYT 41 V_(L)CDR3 QQRSRYPPYT 42 aNRP2-28v2/4 V_(H)CDR1GFSLSTYSIS 63 V_(H)CDR2 IIGDAGGIIYATWAKS 64 V_(H)CDR3 DGTAFDI 65V_(L)CDR1 QASQSIYSKLG 66 V_(L)CDR2 RASTLAS 67 V_(L)CDR3 QQDYSYINVDNI 68

TABLE A2 Exemplary Polypeptide Sequences SEQ ID Description SequenceNO:  aNRP2-1v3 Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWM 43 variableGAIYPGNSDTSYNQQFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC region (V_(H))ARRGGGYFDYWGQGTLVTVSS Light chainDIQLTQSPSFLSASVGDRVTITCKASQNVGAAVAWYQQKPGKAPKLLI 44 variableYSASNRYTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYSSYPL region (V_(L))LTFGGGTKVEIK aNRP2-2v4 Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWM 45 variableGVIHPNSASTFYNERFKTRATMTVDRSSSTAYMELSSLRSEDTAVYYC region (V_(H))SRPGTVRRSDYWGQGTTVTVSS Light chainDVVMTQSPLSLPVTLGQPASISCRSSQNIVHSTGNTYLEWYQQRPGQS 46 variablePRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQG region (V_(L))SHVPWTFGGGTKVEIK aNRP2-10v5 Heavy chainEVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYIHWVQQAPGKGLEWM 47 variableGRIDVEDDETKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYC region (V_(H))ATPIYGSREAWFAYWGQGTLVTVSS Light chainDIQMTQSPSSLSASVGDRVTITCTASSSVSSSYLHWYQQKPGKAPKLL 48 variableIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYRSP region (V_(L))PTFGGGTKVEIK aNRP2-10v10 Heavy chainEVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYVHWVQQAPGKGLEWM 49 variableGRIDVEDDETKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYC region (V_(H))ATPIYGAREAWFAYWGQGTLVTVSS Light chainDIQMTQSPSSLSASVGDRVTITCTASSSVSSSYLHWYQQKPGKAPKLL 50 variableIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYRSP region (V_(L))PTFGGGTKVEIK aNRP2-10v13 Heavy chainEVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYVHWVQQAPGKGLEWM 69 variableGRIDVHDDETKYAPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYC region (V_(H))ATPIYGAREAWFAYWGQGTLVTVSS Light chainDIQMTQSPSSLSASVGDRVTITCTASSSVSSSYLHWYQQKPGKAPKLL 70 variableIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYRSP region (V_(L))PTFGGGTKVEIK aNRP2-11v7 Heavy chainQVQLVQSGAEVKKPGASVKVSCKASGYTFTSFGISWVRQAPGQGLEWI 51 variableGEIYPRSGNTYYNENFKGRATMTADKSTSTAYMELRSLRSDDTAVYYC region (V_(H))ARSSGYYGSTPFPYWGQGTLVTVSS Light chainDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLI 52 variableYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTLPW region (V_(L))TFGGGTKVEIK aNRP2-14v9 Heavy chainQLQLVESGGGVVQPGRSLRLSCTVSGFSLTSYGVHWVRQAPGKGLEWV 53 variableGLIWSGGSTDYSPAFISRFTISEDNSKSTVYLQMNSLRAEDTAVYFCA region (V_(H))RNSYSSGYYAMDYWGQGTTVTVSS Light chainDIQLTQSPSFLSASVGDRVTITCKASQNVGHAVAWYQQKPGKAPKLLI 54 variableYSASNRYTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYSRYPP region (V_(L))YTFGGGTKVEIK aNRP2-14v10 Heavy chainQLQLVESGGGVVQPGRSLRLSCTVSGFSLTSYGVHWVRQAPGKGLEWV 55 variableGLIWSGGSTDYSPAFISRFTISEDNSKSTVYLQMNSLRAEDTAVYFCA region (V_(H))RNSYSSGYYAMDYWGQGTTVTVSS Light chainDIQLTQSPSFLSASVGDRVTITCKASQNVGTAVAWYQQKPGKAPKLLI 56 variableYSASNRYTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQRSRYPP region (V_(L))YTFGGGTKVEIK aNRP2-28v2 Heavy chainQSVKESEGGLFKPTDTLTLTCTVSGFSLSTYSISWVRQAPGNGLEWIG 71 variableIIGDAGGIIYATWAKSRSTITRSTALNTVTLKMTGLTAADTATYFCAR region (V_(H))DGTAFDIWGPGTLVTVSS Light chainAYDMTQTPASVEVVVGGTVTIKCQASQSIYSKLGWYQQKPGQPPKLLI 72 variableYRASTLASGVSSRFKGSGSGTEYTLTISGVQCDDAATYYCQQDYSYIN region (V_(L))VDNIFGGGTEVVVK aNRP2-28v4 Heavy chainQSVKESEGGLFKPTDTLTLTCTVSGFSLSTYSISWVRQAPGNGLEWIG 73 variableIIGDAGGIIYATWAKSRSTITRSTALNTVTLKMTGLTAADTATYFCAR region (V_(H))DGTAFDIWGPGTLVTVSS Light chainAYDMTQTPASVEVVVGGTVTIKCQASQSIYSKLGWYQQKPGQPPKLLI 74 variableYRASTLASGVSSRFKGSGSGTEYTLTISGVQADDAATYYCQQDYSYIN region (V_(L))VDNIFGGGTEVVVK

Thus, in certain embodiments, an antibody or antigen-binding fragmentthereof comprises

-   -   a heavy chain variable region (V_(H)) sequence that comprises        complementary determining region V_(H)CDR1, V_(H)CDR2, and        V_(H)CDR3 sequences selected from Table A1 and variants thereof        which specifically bind to a human NRP2 polypeptide (selected,        for example, from Table N1); and    -   a light chain variable region (V_(L)) sequence that comprises        complementary determining region V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences selected from Table A1 and variants thereof        which specifically bind to the human NRP2 polypeptide (selected,        for example, from Table N1).

In certain embodiments, the CDR sequences are as follows:

-   -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 1-3, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 4-6, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 7-9, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 10-12, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 13-15, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 16-18, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 19-21, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 22-24, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 25-27, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 28-30, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 31-33, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 34-36, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 34-39, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 40-42, respectively,        including variants thereof;    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 57-59, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 60-62, respectively,        including variants thereof; or    -   the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3 sequences comprise SEQ        ID NOs: 63-65, respectively, and the V_(L)CDR1, V_(L)CDR2, and        V_(L)CDR3 sequences comprise SEQ ID NOs: 66-68, respectively,        including variants thereof.

Also included are variants thereof, including affinity matured variants,which bind to human NRP2, for example, variants having 1, 2, 3, 4, 5, or6 alterations in one or more of the CDR regions, for example, one ormore the V_(H)CDR1, V_(H)CDR2, V_(H)CDR3, V_(L)CDR1, V_(L)CDR2, and/orV_(L)CDR3 sequences described herein. Exemplary “alterations” includeamino acid substitutions, additions, and deletions.

In certain embodiments, the V_(H) sequence is at least 80, 85, 90, 95,97, 98, 99, or 100% identical to a sequence selected from Table A2,including, for example, wherein the V_(H) sequence has 1, 2, 3, 4, or 5alterations in one or more framework regions.

In some embodiments, the V_(L) sequence is at least 80, 85, 90, 95, 97,98, 99, or 100% identical to a sequence selected from Table A2,including, for example, wherein the V_(L) sequence has 1, 2, 3, 4, or 5alterations in one or more framework regions.

In some embodiments, the V_(H) and V_(L) sequences of an antibody orantigen-binding fragment are as follows:

-   -   the V_(H) sequence comprises SEQ ID NO: 43, and the V_(L)        sequence comprises SEQ ID NO: 44;    -   the V_(H) sequence comprise SEQ ID NO: 45, and the V_(L)        sequence comprises SEQ ID NO: 46;    -   the V_(H) sequence comprises SEQ ID NO: 47, and the V_(L)        sequence comprises SEQ ID NO: 48;    -   the V_(H) sequence comprises SEQ ID NO: 49, and the V_(L)        sequence comprises SEQ ID NO: 50;    -   the V_(H) sequence comprises SEQ ID NO: 51, and the V_(L)        sequence comprises SEQ ID NO: 52;    -   the V_(H) sequence comprises SEQ ID NO: 53, and the V_(L)        sequence comprises SEQ ID NO: 54;    -   the V_(H) sequence comprises SEQ ID NO: 55, and the V_(L)        sequence comprises SEQ ID NO: 56;    -   the V_(H) sequence comprises SEQ ID NO: 69, and the V_(L)        sequence comprises SEQ ID NO: 70;    -   the V_(H) sequence comprises SEQ ID NO: 71, and the V_(L)        sequence comprise SEQ ID NO: 72; or    -   the V_(H) sequence comprises SEQ ID NO: 73, and the sequence        comprise SEQ ID NO: 74.

Also included are variants thereof, for example, variants having 1, 2,3, 4, or 5 alterations in one or more framework regions. Exemplary“alterations” include amino acid substitutions, additions, anddeletions.

Table A3 below summarizes exemplary redundant antibody CDRs, includingexemplary consensus CDR sequences, and provides amino acid code for thesame.

TABLE A3 Redundant antibody CDRs Antibody Chain CDR1 CDR2 CDR3aNRP2-10 V_(H) GFNX₂₂KDX₄₂X₁₄X₂₂H RIDX₄₃EDDETKYAPKFQGX₁X₂X₃X₄X₅RX₆X₇X₈X₉X₁₀X₁₁ (SEQ ID NO: 75) (SEQ ID NO: 76)(SEQ ID NO: 77) aNRP2-10 _(VL) TASSSVSSSYLH X₂₁TSNLASX₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀ (SEQ ID NO: 16) (SEQ ID NO: 78)(SEQ ID NO: 79) aNRP2-11 V_(H) GYTFTSFGIS EIYPRSGNTYYNENFKGX₂₃X₂₄GX₂₅YX₂₆STPX₂₇X₂₈X₂₉ (SEQ ID NO: 25) (SEQ ID NO: 26)(SEQ ID NO: 80) aNRP2-11 V_(L) RASQDISNYLN YTSRLHS QQGNTLPWT(SEQ ID NO: 28) (SEQ ID NO: 29) (SEQ ID NO: 30) aNRP2-14 V_(H)GFSLTSYGVH LIWSGGSTDYSPAFIS NX₃₀X₃₁X₃₂X₃₃GYYX₃₄X₄₁DX₃₅ (SEQ ID NO: 31)(SEQ ID NO: 32) (SEQ ID NO: 81) aNRP2-14 V_(L) KASQNVGX₄₀AVA SASNRYTQQX₃₆X₃₇X₃₈X₃₉PPYT SEQ ID NO: 82) (SEQ ID NO: 5) (SEQ ID NO: 83)Redundant Amino acid code X₁ AEFGHKLNPTY X₂₃ ST X₂ DEHIKLPQRSTVY X₂₄GKLSTV X₃ AFGHIKLPQRSTVY X₂₅ LY X₄ AFGSTY X₂₆ AGS X₅ AFGHIKNQRSTVY X₂₇AFHLNQSTY X₆ AEPQST X₂₈ AIPTV X₇ AILNQSV X₂₉ AEGHIKLNQRSTY X₈ FWY X₃₀FGISTVY X₉ FL X₃₁ HFY X₁₀ ADFGHKLNPRSTY X₃₂ AFGS X₁₁ ADEFGHIKLNRSTVY X₃₃FSY X₁₂ DFGHIKLNRSTV X₃₄ AGHSPT X₁₃ ADEGQST X₃₅ DGHIAVLRSY X₁₄ FY X₃₆FHKRY X₁₅ FGHNY X₃₇ AILNRSTV X₁₆ HNQRSTY X₃₈ FGIKLNRSTVY X₁₇ AFHILNPSTVX₃₉ AFGIQRSTY X₁₈ ADFGHKLPSTVY X₄₀ AGHNQRST X₁₉ AGLPST X₄₁ MK X₂₀ADEFGHIKLNQRSTVY X₄₂ SY X₂₁ RS X₄₃ VP X₂₂ IV

Thus, in certain embodiments, the at least one anti-NRP2 antibody orantigen-binding fragment thereof comprises a CDR sequence, for example,a CDR1 consensus sequence, from Table A3.

Neuropilin-2 is a cell surface receptor protein that modulates a broadrange of cellular functions through its roles as an essential cellsurface receptor and co-receptor for a variety of ligands (see, e.g.,Guo and Vander Kooi, J. Cell. Biol. 290 No 49: 29120-29126, 2015). Forinstance, it functions during epithelial to mesenchymal transition(EMT), for example, by promoting TGF-β1-mediated EMT in colorectal andother cancer cells (see, e.g., Grandclement et al., PLoS ONE 6(7)e20444, 2011), and by mediating EMT or endo-EMT in fibroblasts,myofibroblasts, and endothelial cells to promote fibrosis formation(see, e.g., Pardali et al., Int. J. Mol. Sci. 18:2157, 2017).

Neuropilin-2 expression promotes lymphangiogenesis (see, e.g., Doci etal., Cancer Res. 75:2937-2948, 2015) single nucleotide polymorphisms(SNPs) in NRP2 are associated with lymphedema (see, e.g., Miaskowski etal., PLoS ONE 8(4) e60164, 2013). NRP2 also regulates smooth musclecontractility (see, e.g., Bielenberg et al., Amer. J. Path. 181:548-559,2012), regulates autophagy, for example, in cancer (see, e.g., Stantonet al., Cancer Res. 73:160-171, 2013), contributes to tumor initiation,survival, and metastasis (see, e.g., Goel et al., EMBO Mol. Med.5:488-508, 2013; and Samuel et al., PLoS ONE 6(10) e23208, 2011), andregulates immune cell activation and migration (see, e.g.,Mendes-da-Cruz et al., PLoS ONE 9(7) e103405, 2014). Neuropilins arealso multifunctional co-receptors involved in tumor initiation, growth,metastasis and immunity (see, e.g., Prud'homme et al., Oncotarget3:921-939, 2012).

Neuropilin-2 is expressed in various cells of the immune system,including lymphoid cells such as B and T cells, and myeloid cells suchas basophils, eosinophil, monocytes, dendritic cells, neutrophils, andmacrophages, including tissue-specific macrophages, for example,alveolar macrophages. It is also expressed in endothelial and epithelialcells in the lung and other tissues, and in muscle cells [see, e.g.,Bielenberg et al., Amer. J. Path. 181:548-559, 2012; Aung, et al., PLoSONE 11(2) e0147358, 2016; Schellenburg et al., Mol. Imm 90:239-244,2017; and Wild et al., Int. J. Exp. Path. 93:81-103, 2012).

Neuropilin-2 also plays a key role in endosome development, for example,by regulating late endosomal maturation, an important aspect ofphagocytosis and efferocytosis, which respectively contribute toclearance of infections and apoptotic cells (See, e.g., Diaz-Vera etal., J. Cell. Sci. 130:697-711, 2017; Dutta et al., Cancer Res.76:418-428, 2016).

Neuropilin-2 is known to be a key player in the pathophysiology of manydiseases (e.g., “NRP2-associated diseases”) and interacts with a broadarray of soluble ligands including semaphorin 3F, VEGF-C and D, andTGF-beta (see, for example, Table N2 and Table N3), and an array ofcellular receptors and co-factors (see, for example, FIGS. 1A-1B andFIG. 2 ). NRP2 is also polysialated on dendritic cells, and activelyinteracts with the chemokine CCL21 to mediate immune cell migration, andfor which single nucleotide polymorphisms associated with ILD and RAhave been described (see, e.g., Rey-Gallardo et al., Glycobiology20:1139-1146, 2010; Stahl et al., Nat. Genet. 42:508-514, 2013; andMiller et al., Arthritis Rheum. 65:3239-3247). Additionally, soluble,circulating forms of NRP-2 are known (see, e.g., Parker et al.,Structure 23(4) 677-687, 2015), and internal studies have confirmed theexistence of circulating complexes of HRS polypeptides and NRP-2polypeptides in circulation. Accordingly, given the central role playedby NRP2 in pathophysiology in a broad range of diseases, it is evidentthat interactions between NRP2 and NRP2 ligand(s) (for example, NRP2ligands from Table N2 and Table N3), and the modulation of thoseinteractions with antibodies against NRP2 to selectively change thecorresponding biological activities, provides broad potential for thetreatment of diseases, including NRP2 associated diseases.

NRP2 is a single transmembrane receptor with a predominant extracellularregion containing two CUB domains (a1/a2 combined domain), two FactorV/VIII homology domains (b1/b2 combined domain), a MAM domain (c domain)(see FIGS. 1A-1B), and a short juxtamembrane region that connects the cdomain to the transmembrane domain (which traverses the plasm membrane).The a1a2 combined domain interacts with sema region of the semaphorins,and the b1 domain interacts with the semaphorin PSI and Ig-like domains.NRP2 has a higher affinity for SEMA3F and 3G; in contrast, SEMAs 3A, 3Band 3E preferentially interact with NRP1. Both NRP1 and NRP2 havesimilar affinity for SEMA 3C. The b1b2 combined domain interacts withseveral growth factors containing heparin-binding domains, includingVEGF C & D, placenta growth factor (PIGF)-2, fibroblast growth factor(FGF), galectin, hepatocyte growth factor (HGF), platelet derived growthfactor (PDGF), and transforming growth factor (TGF)-beta (see, forexample, Prud'homme et al., Oncotarget. 3:921-939, 2012). NRP2 alsointeracts with various growth factor-specific receptors, andinteractions with these receptors occur independently of binding toSEMAs. In this context, integrins and growth factor receptors like VEGFreceptor, TGF-beta receptor, c-Met, EGFR, FGFR, PDGFR, have been shownto interact with NRPs and in general appear to increase the affinity ofeach ligand for its receptor and to modulate down stream signaling. Thec domain (Mam) domain does not appear to be directly required for ligandbinding, but may impact ligand specificity, receptor signaling, and NRP2dimerization. The juxtamembrane region differs significantly between theNRP2a and NRP2b isoforms, and may also impact ligand specificity,dimerization, and signaling.

Accordingly, anti-NRP2 antibodies and antigen-binding fragments thereofthat bind to the a1 and/or a2 domains of NRP2 have the potential toselectively modulate semaphorin binding. Likewise, anti-NRP2 antibodiesand antigen-binding fragments thereof that bind to the b1 domain havethe potential to modulating both semaphorin and VEGF and growth factorbinding, and anti-NRP2 antibodies that bind to the b2 domain have thepotential to selectively modulate VEGF and growth factor binding.Antibodies and antigen-binding fragments thereof that bind to the cdomain might not directly impact NRP2 ligand binding, but have thepotential to modulate NRP2 downstream signaling, for example, bymodulating (e.g., promoting or enhancing) NRP2 receptor dimerization.

Anti-NRP2 antibodies and antigen binding fragments thereof that promoteNRP2 receptor homodimerization could modulate NRP2 receptor activity,and provide agonistic or antagonistic antibodies depending on the natureof the binding site. Such antibodies and antigen binding fragmentsthereof could modulate (e.g., enhance) the activities of NRP2 ligands,even if they do not directly modulate ligand binding. Additionaldiversity in the functional effects of specific anti-NRP2 antibodies maybe expected based on their binding mode, and as a result of stericeffects, which may indirectly impact ligand binding.

NRP2 can form homodimers as well as heterodimers, and is heavilyglycosylated. NRP2 has different splice variants which are between about551 and 926 amino acids long. Two major variants for NRP2 arecategorized as NRP2a and NRP2b. These differ in their intracellular Cterminal part (FIGS. 1A-1B) in which for NRP2a, the c-terminal domaincomprises 42 amino acids and a PDZ-binding domain with the C-terminalSEA amino acid sequence. By contrast, NRP2b comprises a 46 amino acid Cterminal domain which shares about 11% sequence homolog with theintracellular, juxtamembrane, and transmembrane sequences of NRP2a.Between the MAM domain and the transmembrane domain, additional splicingcan occur and 5 additional amino acids (GENFK; SEQ ID NO: 153) can beadded to either the NRP2a, or NRP2b forms—these variants are named basedon the number additional amino acids added through alternative splicing.Thus the two variants of NRP2a are named NRP2a(17) (or variant 1) andNRP2a(22) (or variant 2), and the two transmembrane variants for NRP2bare named NRP2b(0) (or variant 4) and NRP2b(5) (or variant 5).Additionally, a soluble form called sNRP2b (or variant 6) can begenerated. Exemplary NRP2 polypeptide sequences are provided in Table N1below, including the mature (after cleavage of the N-terminal signalpeptide) and precursor forms of the various isoforms of NRP2.

TABLE N1 Exemplary Human NRP2 Polypeptides SEQ ID Name Residues SequenceNO: Human full   1-931 MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYI 84length NRP2 TSPGYPQDYPSHQNCEWIVYAPEPNQKIVLNFNPHFEIEK Variant 1HDCKYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSML precursorYIRFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTI NRP2a(22)ESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAGENFKVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYTLDPILITIIAMSSLGVLLGATCAGLLLYCTCSYSGLSSRSCTTLENYNFELYDGLKHKV KMNHQKCCSEA Human NRP2   1-926MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYI 85 Variant 2TSPGYPQDYPSHQNCEWIVYAPEPNQKIVLNFNPHFEIEK precursorHDCKYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSML NRP2a(17)YIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYTLDPILITIIAMSSLGVLLGATCAGLLLYCTCSYSGLSSRSCTTLENYNFELYDGLKHKVKMNHQ KCCSEA Human NRP2   1-909MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYI 86 Variant 3TSPGYPQDYPSHQNCEWIVYAPEPNQKIVLNFNPHFEIEK precursorHDCKYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSML NRP2a(0)YIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFADEYEVDWSNSSSATSGSGAPSTDKEKSWLYTLDPILITIIAMSSLGVLLGATCAGLLLYCTCSYSGLSSRSC TTLENYNFELYDGLKHKVKMNHQKCCSEAHuman NRP2   1-906 MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYI 87 Variant 4TSPGYPQDYPSHQNCEWIVYAPEPNQKIVLNFNPHFEIEK precursorHDCKYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSML NRP2b(5)YIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAGENFKGGTLLPGTEPTVDTVPMQPIPAYWYYVMAAGGAVLVLVSVALALVLHYHRFRYAAKKTDHSITYKTS HYTNGAPLAVEPTLTIKLEQDRGSHCHuman NRP2   1-901 MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYI 88 Variant 5TSPGYPQDYPSHQNCEWIVYAPEPNQKIVLNFNPHFEIEK precursorHDCKYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSML NRP2b(0)YIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAGGTLLPGTEPTVDTVPMQPIPAYWYYVMAAGGAVLVLVSVALALVLHYHRFRYAAKKTDHSITYKTSHYTNG APLAVEPTLTIKLEQDRGSHCHuman NRP2   1-555 MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYI 89 Variant 6TSPGYPQDYPSHQNCEWIVYAPEPNQKIVLNFNPHFEIEK precursorHDCKYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSML S9NRP2bYIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTI Soluble NRP2ESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKVGCSWRPL Human NRP2  23-926QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAP 90 Variant 2EPNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLG NRP2a(17)KHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIF (mature)KTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTREVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYTLDPILITIIAMSSLGVLLGATCAGLLLYCTCSYSGLSSRSCTTLEN YNFELYDGLKHKVKMNHQKCCSEANRP2 splice  23-901 QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAP 91variant 5 EPNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLG NRP2b(0)KHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIF (mature)KTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTREVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAGGTLLPGTEPTVDTVPMQPIPAYWYYVMAAGGAVLVLVSVALALVLHYHRFRYAAKKTDHSITYKTSHYTNGAPLAVEPTLTIKLEQDRGSHC Soluble NRP2  23-555QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAP 92 S9Nrp-2bEPNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLG (mature)KHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTREVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPR TQQPKVGCSWRPL NRP2 a1 domain 28-141 CGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAPEPNQK 93IVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEI NRP2 a2 domain 149-265CSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEII 94LQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYY NRP2 b1 domain 280-426PLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWT 95PNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLH APLLTRFVRIRPQTWHSGIALRLELFGNRP2 b2 domain 438-591 LGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPR 96IPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLG NRP2 c domain 641-794PSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTF 97PDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTD NRP2  23-265QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAP 98 a1a2 combinedEPNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLG domainsKHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSA RYY NRP2 149-426CSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEII 99 a2b1 combinedLQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGK domainsYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFG NRP2  23-426QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAP 100 a1a2b1EPNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLG combinedKHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIF domainsKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRL ELFG NRP2  23-595QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAP 101 a1a2b1b2EPNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLG combinedKHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIF domainsKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAG IGMRLEVLGCDWT NRP2 a2b1b2145-595 GSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPK 102 combinedMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGP domainsLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIG MRLEVLGCDWT NRP2 276-595QCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDD 103 b1b2 combinedNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNG domainsYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWT NRP2 v2 - Fc  23-855QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAP 104 fusion proteinEPNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKNRP2 a2b1b2-Fc 145-595 GSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPK 105MEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK NRP2 438-794LGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPR 106 b2c combinedIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEA domainsRAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTD NRP2 276-794QCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDD 107 b1b2c combinedNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNG domainsYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTD NRP2 149-802CSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEII 108 a2b1b2cLQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGK combinedYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVH domainsQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDD IRISTDVPLENCME NRP2  23-802QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAP 109 a1a2b1b2cEPNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLG combinedKHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIF domainsKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSG EIAIDDIRISTDVPLENCME NRP2a 23-859 QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAP 110 a1a2b1b2cEPNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLG combinedKHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIF domains +KTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAK juxtamembranePKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYTLDP NRP2bQPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAP 111 a1a2b1b2cEPNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLG combinedKHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIF domains +KTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAK juxtamembranePKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCSFEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLWVIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFAGGTLLPGTEPTVDT VPMQPIPAY NRP2a 803-864PISAFAGENFKVDIPEIHEREGYEDEIDDEYEVDWSNSSS 112 JuxtamembraneATSGSGAPSTDKEKSWLYTLDP Variant 1 NRP2a 803-859PISAFAVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGS 113 JuxtamembraneGAPSTDKEKSWLYTLDP Variant 2 NRP2a 803-842PISAFADEYEVDWSNSSSATSGSGAPSTDKEKSWLYTLDP 114 Juxtamembrane Variant 3NRP2b 803-837 PISAFAGENFKGGTLLPGTEPTVDTVPMQPIPAYW 115 JuxtamembraneVariant 4 NRP2b 803-832 PISAFAGGTLLPGTEPTVDTVPMQPIPAYW 116 JuxtamembraneVariant 5 NRP2 QPDPPCGGRLNSKDAGYITSPGYPQDYPSHQNCEWIVYAP 117 (23-595)-FcEPNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCGNIAPPTIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPHNLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYCGTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRIANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQGAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLHAPLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEYLWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVEARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRVYPERWSPAGIGMRLEVLGCDWTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In certain embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to a full-length human NRP2polypeptide or a human NRP2 polypeptide selected from Table N1. In someembodiments, the antibody or antigen-binding fragment thereof binds tothe human NRP2 polypeptide with an affinity of about 10 pM to about 500pM or to about 50 nM, or about, at least about, or no more than about10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM,25 nM, or 50 nM, or optionally with an affinity that ranges from about10 pM to about 500 pM, about 10 pM to about 400 pM, about 10 pM to about300 pM, about 10 pM to about 200 pM, about 10 pM to about 100 pM, about10 pM to about 50 pM, or about 20 pM to about 500 pM, about 20 pM toabout 400 pM, about 20 pM to about 300 pM, about 20 pM to about 200 pM,about 20 pM to about 100 pM, about 20 pM to about 50 pM, or about 30 pMto about 500 pM, about 30 pM to about 400 pM, about 30 pM to about 300pM, about 30 pM to about 200 pM, about 30 pM to about 100 pM, about 30pM to about 50 pM, or about 20 pM to about 200 pM, about 30 pM to about300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 1nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, orabout 25 nM to about 50 nM.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof that specifically binds to at least one epitope in atleast one neuropilin domain. Exemplary neuropilin domains include one ormore of the neuropilin a1 domain, neuropilin a2 domain, neuropilin b1domain, neuropilin b2 domain, neuropilin c domain, neuropilin a1/a2combined domain, neuropilin b1/b2 combined domain, neuropilin a2/b1combined domain, neuropilin b2/c combined domain, neuropilin a2/b1/b2combined domain, neuropilin a2/b1/b2/c combined domain, neuropilina1/a2/b1 combined domain, neuropilin a1/a2/b1/b2 combined domain,neuropilin a1/a2/b1/b2/c combined domain, a1/a2/b1/b2/c/juxtamembranecombined domain, and the neuropilin b1/b2/c combined domain (see TableN1 for residues of the domains). In specific embodiments, the at leastone antibody or antigen-binding fragment thereof specifically binds toat least one epitope in the neuropilin b1 domain, the neuropilin b2domain, and/or the neuropilin b1/b2 combined domain (see Table N1). Inparticular embodiments, the antibody or antigen-binding fragment thereofbinds to the at least one domain (or at least one epitope therein) withan affinity of about 10 pM to about 500 pM or to about 50 nM, or about,at least about, or no more than about 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400,500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM, or optionallywith an affinity that ranges from about 10 pM to about 500 pM, about 10pM to about 400 pM, about 10 pM to about 300 pM, about 10 pM to about200 pM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, orabout 20 pM to about 500 pM, about 20 pM to about 400 pM, about 20 pM toabout 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM,about 20 pM to about 50 pM, or about 30 pM to about 500 pM, about 30 pMto about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or about 20pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about900 pM, about 100 pM to about 1 nM, about 1 nM to about 5 nM, about 5 nMto about 10 nM, about 10 nM to 25 nM, or about 25 nM to about 50 nM.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin a1 domain, the neuropilin a2 domain, and/or the neuropilina1a2 combined domain, including adjacent linker regions, for example, atabout residues (neuropilin a1 domain) 20-148, 30-141, 40-141, 50-141,60-141, 70-141, 80-141, 90-141, 100-141, 110-141, 120-141, 130-141;20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50,20-40, or 20-30 as defined by a human NRP2 precursor sequence (see TableN1); or, for example, at about residues (neuropilin a2 domain) 142-280,150-265, 160-265, 170-265, 180-265, 190-265, 200-265, 210-265, 220-265,230-265, 240-265, 250-265, 260-265, 141-270, 141-260, 141-250, 141-240,141-230, 141-220, 141-210, 141-200, 141-190, 141-180, 141-170, 141-160,141-150, 200-250, 210-250, 220-250, 230-250, 200-240, 210-240, 220-240,230-240, 227-247, 228-247, 229-247, 230-247, 231-247, 232-247, 233-247,234-247, 235-247, 236-247; 227-246, 227-245, 227-244, 227-243, 227-242,227-241, 227-240, 227-239, 227-238; 235-240, 236-239, 236-238, orresidue 237 as defined by a human NRP2 precursor sequence (see TableN1); or, for example, at about residues (combined a1a2 domain) 20-280,30-280, 40-280, 50-280, 60-280, 70-280, 80-280, 90-280, 100-280,110-280, 120-280, 130-280, 140-280, 150-280, 160-280, 170-280, 180-280,190-280, 200-280, 210-280, 220-280, 230-280, 240-280, 260-280, 270-280,20-270, 20-260, 20-250, 20-240, 20-230, 20-220, 20-210, 20-200, 20-190,20-180, 20-170, 20-160, 20-150, 20-140, 20-130, 20-120, 20-110, 20-100,20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30 as defined by a humanNRP2 precursor sequence (see Table N1).

In particular embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin b1 domain, the neuropilin b2 domain, and/or the neuropilinb1/b2 combined domain, including adjacent linker regions, for example,at about residues (neuropilin b1 domain) 266-426, 280-426, 290-426,299-420, 300-426, 310-426, 320-426, 330-426, 340-426, 350-426, 360-426,370-426, 380-426, 390-426, 400-426, 410-426, 420-426, 280-420, 280-410,280-400, 280-390, 280-380, 280-370, 280-360, 280-350, 280-340, 280-330,280-320, 280-310, 280-300, or 280-290 as defined by a human NRP2precursor sequence (see Table N1), including a discontinuous epitopethat comprises one, two, or three of residues Y299, N354, and/or S416 asdefined by the human NRP2 precursor sequence; (neuropilin b2 domain)438-591, 450-591, 460-591, 470-591, 480-591, 490-591, 500-591, 510-591,520-591, 530-591, 540-591, 550-591, 560-591, 570-591, 580-591, 438-590,438-580, 438-570, 438-560, 438-550, 438-540, 438-530, 438-520, 438-510,438-500, 438-490, 438-480, 438-470, 438-460, 438-450 as defined by ahuman NRP2 precursor sequence (see Table N1); or (neuropilin b1/b2combined domain) 266-591, 276-591, 286-591, 296-591, 306-591, 316-591,326-591, 336-591, 346-591, 356-591, 366-591, 376-591, 386-591, 396-591,406-591, 416-591, 426-591, 436-591, 446-591, 456-591, 466-591, 476-591,486-591, 498-591, 508-591, 518-591, 528-591, 538-591, 548-591, 558-591,568-591, 578-591, 588-591, 266-581, 266-571, 266-561, 266-551, 266-541,266-531, 266-521, 266-511, 266-501, 266-491, 266-481, 266-471, 266-461,266-451, 266-441, 266-431, 266-421, 266-411, 266-401, 266-391, 266-381,266-371, 266-361, 266-351, 266-341, 266-331, 266-321, 266-311, 266-301,266-291, 266-281, or 266-271 as defined by a human NRP2 precursorsequence (see Table N1).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin a2/b1 combined domain and/or the neuropilin b2c combineddomain, including adjacent linker regions, for example, at aboutresidues (neuropilin a2b1 combined domain) 149-437, 159-426, 169-426,179-426, 189-426, 199-426, 209-426, 219-426, 229-426, 239-426, 249-426,259-426, 269-426, 279-426, 289-426, 299-426, 309-426, 319-426, 329-426,339-426, 349-426, 359-426, 369-426, 379-426, 389-426, 399-426, 409-426,419-426, 149-436, 149-426, 149-416, 149-406, 149-396, 149-386, 149-376,149-366, 149-356, 149-346, 149-336, 149-326, 149-316, 149-306, 149-296,149-286, 149-276, 149-266, 149-256, 149-246, 149-236, 149-226, 149-216,149-206, 149-196, 146-186, 146-176, 146-166, or 146-155 as defined by ahuman NRP2 precursor sequence (see Table N1); or, for example, at aboutresidues (neuropilin b2c combined domain) 438-794, 448-794, 458-794,468-794, 478-794, 487-794, 497-794, 507-794, 517-794, 527-794, 537-794,547-794, 557-794, 567-794, 587-794, 597-794, 607-794, 617-794, 627-794,637-794, 647-794, 657-794, 667-794, 677-794, 687-794, 697-794, 707-794,717-794, 727-794, 737-794, 747-794, 757-794, 767-794, 777-794, 787-794,427-794, 438-784, 438-774, 438-764, 438-754, 438-744, 438-734, 438-728,438-714, 438-704, 438-694, 438-684, 438-674, 438-664, 438-654, 438-644,438-634, 438-624, 438-614, 438-604, 438-596, 438-586, 438-576, 438-566,438-556, 438-546, 438-536, 438-526, 438-516, 438-506, 438-494, 438-484,438-474, 438-464, 438-454, 438-444 as defined by a human NRP2 precursorsequence (see Table N1).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin c domain, including adjacent linker regions, for example, atabout residues 591-794, 600-794, 610-794, 620-794, 630-794, 640-794,650-794, 660-794, 670-794, 680-794, 690-794, 700-794, 710-794, 720-794,730-794, 740-794, 750-794, 760-794, 770-794, 780-794, 790-794, 591-790,591-780, 591-770, 591-760, 591-750, 591-740, 591-730, 591-720, 591-710,591-700, 591-690, 591-680, 591-670, 591-660, 591-650, 591-640, 591-630,591-620, 591-610, or 591-600 as defined by a human NRP2 precursorsequence (see Table N1).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin b1/b2/c combined domain, including adjacent linker regions,for example, at about residues 276-794, 286-794, 296-794, 306-794,316-794, 326-794, 336-794, 346-794, 356-794, 366-794, 376-794, 387-794,396-794, 406-794, 416-794, 426-794, 436-794, 446-794, 456-794, 466-794,476-794, 486-794, 496-794, 506-794, 516-794, 526-794, 536-794, 546-794,556-794, 566-794, 576-794, 586-794, 596-794, 606-794, 616-794, 626-794,636-794, 646-794, 656-794, 666-794, 676-794, 686-794, 696-794, 706-794,716-794, 726-794, 736-794, 746-794, 756-794, 766-794, 776-794, 786-794,266-794, 276-784, 276-774, 276-764, 276-754, 276-744, 276-734, 276-724,276-714, 276-704, 276-694, 276-684, 276-674, 276-664, 276-654, 276-644,276-634, 276-624, 276-614, 276-604, 276-594, 276-584, 276-574, 276-564,276-554, 276-544, 276-534, 276-524, 276-514, 276-504, 276-594, 276-584,276-574, 276-564, 276-554, 276-544, 276-534, 276-524, 276-514, 276-504,or 276-496 as defined by a human NRP2 precursor sequence (see Table N1).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin C/juxtamembrane combined domain.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to at least one epitope in theneuropilin juxtamembrane domain of NRP2a (variant 1). In someembodiments, the at least one antibody or antigen-binding fragmentthereof specifically binds to at least one epitope in the neuropilinjuxtamembrane domain of NRP2a (variant 2). In some embodiments, the atleast one antibody or antigen-binding fragment thereof specificallybinds to at least one epitope in the neuropilin juxtamembrane domain ofNRP2a (variant 3). In some embodiments, the at least one antibody orantigen-binding fragment thereof specifically binds to at least oneepitope in the neuropilin juxtamembrane domain of NRP2b (variant 4). Insome embodiments, the at least one antibody or antigen-binding fragmentthereof specifically binds to at least one epitope in the neuropilinjuxtamembrane domain of NRP2b (variant 5).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to a conformational epitope composedof two or more discontinuous epitope regions. In some embodiments, theat least one antibody or antigen-binding fragment thereof specificallybinds to a conformational epitope comprising or consisting of:

-   -   (a) a first epitope region within the a1 domain, and second        epitope region within the a2 domain of the human NPR2        polypeptide;    -   (b) a first epitope region within the a1 domain, and second        epitope region within the b1 domain of the human NPR2        polypeptide;    -   (c) a first epitope region within the a1 domain, and second        epitope region within the b2 domain of the human NPR2        polypeptide;    -   (d) a first epitope region within the a1 domain, and second        epitope region within the c domain of the human NPR2        polypeptide;    -   (e) a first epitope region within the a1 domain, and second        epitope region within the juxtamembrane domain of the human NPR2        polypeptide selected from variant 1, 2, 3, 4 and 5;    -   (f) a first epitope region within the a2 domain, and second        epitope region within the b1 domain of the human NPR2        polypeptide;    -   (g) a first epitope region within the a2 domain, and second        epitope region within the b2 domain of the human NPR2        polypeptide;    -   (h) a first epitope region within the a2 domain, and second        epitope region within the c domain of the human NPR2        polypeptide;    -   (i) a first epitope region within the a2 domain, and second        epitope region within the juxtamembrane domain of the human NPR2        polypeptide selected from variant 1, 2, 3, 4 and 5;    -   (j) a first epitope region within the b1 domain, and second        epitope region within the b2 domain of the human NPR2        polypeptide;    -   (k) a first epitope region within the b1 domain, and second        epitope region within the c domain of the human NPR2        polypeptide;    -   (l) a first epitope region within the b1 domain, and second        epitope region within the juxtamembrane domain of the human NPR2        polypeptide selected from variant 1, 2, 3, 4 and 5;    -   (m) a first epitope region within the b2 domain, and second        epitope region within the c domain of the human NPR2        polypeptide;    -   (n) a first epitope region within the b2 domain, and second        epitope region within the juxtamembrane domain of the human NPR2        polypeptide selected from variant 1, 2, 3, 4 and 5; or    -   (o) a first epitope region within the c domain, and second        epitope region within the juxtamembrane domain of the human NPR2        polypeptide selected from variant 1, 2, 3, 4 and 5.

In some embodiments, the antibody or antigen-binding fragment thereofspecifically binds to at least one epitope within a region of a humanNRP2 polypeptide that binds to or interacts with at least one “NRP2ligand”, including any molecule that interacts with or binds reversiblyto human NRP2, including any one or more variants of human NRP2. Generalexamples of “NRP2 ligands” include polypeptides such as HRSpolypeptides, soluble ligands, receptors (e.g., cell surface receptors),including growth factors, growth factor receptors, and others, andspecific examples of NRP2 ligands are detailed herein. In someembodiments, the at least one antibody or antigen-binding fragmentthereof modulates (e.g., antagonizes, interferes with, agonizes,enhances) binding of the human NRP2 polypeptide to at least one “NRP2ligand”.

As noted above, in certain embodiments the at least one NRP2 ligand isan HRS polypeptide. Thus, in certain embodiments, an antibody orantigen-binding fragment thereof specifically binds to at least oneepitope within a region of a human NRP2 polypeptide that binds to orinteracts with at least one human HRS polypeptide, and thereby modulatesbinding of the human NRP2 polypeptide to the human HRS polypeptide.Exemplary HRS polypeptides are provided in Table H1 below.

TABLE H1 Exemplary Human HRS polypeptides SEQ ID Name Residues SequenceNO:  FL   1-509 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 118 cytosolicKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIR wild typeCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRRE DLVEEIKRRTGQPLCIC HisRS1^(N1)  1-141 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 119KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGE LLSLRYDLTVPFARYLAM HisRS1^(N2)  1-408 MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 120KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTE HisRS1^(N3)   1-113MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 121KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKL HisRS1^(N4)  1-60MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 122 KAQLGPDESKQKFVLKTPKHisRS1^(N5) 1-243 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 123 27aaKAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVGY PWWNSCSRILNYPKTSRPWRAWETHisRS1^(C1) 405-509 RTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKN 124PKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREE VDVRREDLVEEIKRRTGQPLCICHisRS1^(C2) 1-60 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 125 175-509KAQLGPDESKQKFVLKTPKDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTS REEVDVRREDLVEEIKRRTGQPLCICHisRS1^(C3) 1-60 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 126 211-509KAQLGPDESKQKFVLKTPKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQPLCIC HisRS1^(C4) 1-100 +MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 127 211-509KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLR SVTSREEVDVRREDLVEEIKRRTGQPLCICHisRS1^(C5) 1-174 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 128211-509 KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEV DVRREDLVEEIKRRTGQPLCICHisRS1^(C6) 1-60 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 129 101-509KAQLGPDESKQKFVLKTPKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRR EDLVEEIKRRTGQPLCIC HisRS1^(C7)1-100 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 130 175-509KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSR EEVDVRREDLVEEIKRRTGQPLCICHisRS1^(C8) 1-60 + MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 131 399-509KAQLGPDESKQKFVLKTPKALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRT GQPLCIC HisRS1^(C9) 1-100 +MAERAALEELVKLQGERVRGLKQQKASAELIEEEVAKLLKL 132 399-509KAQLGPDESKQKFVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTG QPLCIC HisRS1^(C10) 369-509MFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTET 133QVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRR EDLVEEIKRRTGQPLCIC HisRS1^(I1)191-333 CLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKF 134RTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFG IDDKISFDLSLARGLDYYTG FL mito.  1-506 MPLLGLLPRRAWASLLSQLLRPPCASCTGAVRCQSQVAEAV 135 wild typeLTSQLKAHQEKPNFIIKTPKGTRDLSPQHMVVREKILDLVISCFKRHGAKGMDTPAFELKETLTEKYGEDSGLMYDLKDQGGELLSLRYDLTVPFARYLAMNKVKKMKRYHVGKVWRRESPTIVQGRYREFCQCDFDIAGQFDPMIPDAECLKIMCEILSGLQLGDFLIKVNDRRIVDGMFAVCGVPESKFRAICSSIDKLDKMAWKDVRHEMVVKKGLAPEVADRIGDYVQCHGGVSLVEQMFQDPRLSQNKQALEGLGDLKLLFEYLTLFGIADKISFDLSLARGLDYYTGVIYEAVLLQTPTQAGEEPLNVGSVAAGGRYDGLVGMFDPKGHKVPCVGLSIGVERIFYIVEQRMKTKGEKVRTTETQVFVATPQKNFLQERLKLIAELWDSGIKAEMLYKNNPKLLTQLHYCESTGIPLVVIIGEQELKEGVIKIRSVASREEVAIKR ENFVAEIQKRLSES 152-398HVGKVWRRESPTIVQGRYREFCQCDFDIAGQFDPMIPDAEC 136LKIMCEILSGLQLGDFLIKVNDRRIVDGMFAVCGVPESKFRAICSSIDKLDKMAWKDVRHEMVVKKGLAPEVADRIGDYVQCHGGVSLVEQMFQDPRLSQNKQALEGLGDLKLLFEYLTLFGIADKISFDLSLARGLDYYTGVIYEAVLLQTPTQAGEEPLNVGSVAAGGRYDGLVGMFDPKGHKVPCVGLSIGVERIFYIVEQR M 294-372QALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGV 137IYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDP Amino-  54-509FVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDT 138 acylationPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPF domain andARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDF anticodonDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRIL bindingDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKG domainLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVERIFSIVEQRLEALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEVDVRREDLVEEIKRRTGQ PLCIC Amino-  54-398FVLKTPKGTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDT 139 acylationPVFELKETLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPF domainARYLAMNKLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFDPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVG LSIGVERIFSIVEQRLE Amino- 61-398 GTRDYSPRQMAVREKVFDVIIRCFKRHGAEVIDTPVFELKE 140 acylationTLMGKYGEDSKLIYDLKDQGGELLSLRYDLTVPFARYLAMN (core)KLTNIKRYHIAKVYRRDNPAMTRGRYREFYQCDFDIAGNFD domainPMIPDAECLKIMCEILSSLQIGDFLVKVNDRRILDGMFAICGVSDSKFRTICSSVDKLDKVSWEEVKNEMVGEKGLAPEVADRIGDYVQQHGGVSLVEQLLQDPKLSQNKQALEGLGDLKLLFEYLTLFGIDDKISFDLSLARGLDYYTGVIYEAVLLQTPAQAGEEPLGVGSVAAGGRYDGLVGMFDPKGRKVPCVGLSIGVER IFSIVEQRLE Anticodon 399-509ALEEKIRTTETQVLVASAQKKLLEERLKLVSELWDAGIKAE 141 bindingLLYKKNPKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRS domainVTSREEVDVRREDLVEEIKRRTGQPLCIC Anticodon 406-501TTETQVLVASAQKKLLEERLKLVSELWDAGIKAELLYKKNP 142 bindingKLLNQLQYCEEAGIPLVAIIGEQELKDGVIKLRSVTSREEV (core) DVRREDLVEEIKRR domainHRS WHEP X_(A)-L-X_(B)-Q-G-X-X-V-R-X-L-K-X-X-K-A-X_(C)-V-X- 143consensus X-L-L-X-L-K-X_(D) Where: X is any amino acidX_(A )is 0-50 amino acids X_(B )is about 5-7 amino acids,preferably 6 amino acids X_(C )is about 7-9 amino acids,preferably 8 amino acids X_(D )is 0-50 amino acids

Thus, in certain embodiments, the at least one NRP2 ligand is selectedfrom Table H1, and the anti-NRP2 antibody or antigen-binding fragmentthereof modulates (e.g., interferes with) binding of a human NRP2polypeptide (for example, a human NRP2 polypeptide selected from TableN1) to a human HRS polypeptide selected from Table H1. In someembodiments, the anti-NRP2 antibody or antigen-binding fragmentspecifically binds to an HRS polypeptide-interacting region of the NRP2polypeptide, and in some instances mimics one or more signalingactivities of the HRS polypeptide binding to the NRP2 polypeptide, forexample, as an agonist antibody. An “HRS polypeptide-interacting region”includes a region or domain of a human NRP2 polypeptide that interactswith a region or domain of human HRS polypeptide, for example, at aligand binding site for a different NRP2 ligand (examples of which areprovided herein), a dimerization domain, a protein-protein interactiondomain, or at a site which is allosterically sensitive within a NRP2polypeptide to modulate the activity of the NRP2 polypeptide.

In certain embodiments, an antibody or antigen-binding fragment thereofis a “blocking antibody”, which fully or substantially inhibits thebinding between a human NRP2 polypeptide (selected, for example, fromTable N1) and an NRP2 ligand such as a human HRS polypeptide (selected,for example, from Table H1) or other NRP2 ligand (for example, selectedfrom Table N2 or Table N3). In some embodiments, a “blocking antibody”inhibits about or at least about 80-100% (e.g., 80, 85, 90, 95, or 100%)of the theoretical maximal binding between the NRP2 polypeptide and theNRP2 ligand (for example, HRS polypeptide) after pre-incubation of the“blocking antibody” with the NRP2 polypeptide in a substantiallystoichiometrically equivalent amount. As used herein, a“stoichiometrically equivalent amount” refers to a situation where thenumber of moles of one substance (e.g., anti-NRP2 antibody) isequivalent or substantially equivalent to the number of moles at leastone other substance (e.g., NRP2 polypeptide) in a given equation orreaction.

In certain embodiments, an antibody or antigen-binding fragment thereofis a “partial-blocking antibody”, which at least partially but not fullyinhibits the binding between a human NRP2 polypeptide (selected, forexample, from Table N1) and an NRP2 ligand such as a human HRSpolypeptide (selected, for example, from Table H1) or other NRP2 ligand(for example, selected from Table N2 or Table N3). In some embodiments,a “partial-blocking antibody” inhibits about or at least about 20-80%(e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80%) of thetheoretical maximal binding between the NRP2 polypeptide and the NRP2ligand (for example, HRS polypeptide) after pre-incubation of the“partial-blocking antibody” with the NRP2 polypeptide in astoichiometric amount.

In specific embodiments, the at least one antibody or antigen-bindingfragment thereof specifically inhibits or otherwise reduces the bindingbetween a human NRP2 polypeptide and a HRS polypeptide splice variantselected from Table H1, for example, a HRS splice variant selected fromone or more of HisRS^(N1), HisRS^(N2), HisRS^(N3), HisRS^(N4) (SV9),HisRS^(N5), HisRS^(C1), HisRS^(C2), HisRS^(C3), HisRS^(C4), HisRS^(C5),HisRS^(C6), HisRS^(C7), HisRS^(C8) (SV11), and HisRS^(C9) (SV14).

As noted above, NRP2 interacts with multiple NRP2 ligands other thanHRS, which mediate downstream signaling events. Additional examples ofNRP2 ligands are provided in Table N2 and Table N3 below.

TABLE N2 Exemplary Neuropilin Ligands Ligand NRP1 NRP2 VEGF-A121 +VEGF-A145 + VEGF-A165 + + VEGF-B167 + VEGF-C + + VEGF-D + + VEGF-E +PIGF-2 + + VEGFR +R1 and R2 +R1, R2, R3 Heparin + + Sema3A + Sema3B, C,D, F, and G + + Plexins A1, A2, A3, A4, D1 + + GIPC1, 2, and 3 + +TGF-β1, β2, and β3 receptors, and LAP + + TbRI and TbRII + + FGF-1, 2,4, and 7 + + FGF receptor 1 + + Hepatocyte growth factor receptors +Integrins (see Table N3) + + Fibronectin + Galectin-1 and GalectinReceptors + + Li-CAM + + Glat-1 + HRS polypeptides (see Table H1) +

TABLE N3 Vertebrate integrins as NRP2 ligands Name Synonyms DistributionLigands α₁β₁ VLA-1 Many Collagens, laminins α₂β₁ VLA-2 Many Collagens,laminins α₃β₁ VLA-3 Many Laminin-5 α₄β₁ VLA-4 Hematopoietic cellsFibronectin, VCAM-1 α₅β₁ VLA-5; widespread fibronectin and proteinasesfibronectin receptor α₆β₁ VLA-6; widespread laminins laminin receptorα₇β₁ muscle, glioma laminins α_(L)β₂ LFA-1 T-lymphocytes ICAM-1, ICAM-2α_(M)β₂ Mac-1, CR3 Neutrophils and monocytes Serum proteins, ICAM-1α_(IIb)β₃ Fibrinogen Platelets fibrinogen, fibronectin^([24]) receptor;gpIIbIIIa α_(V)β₁ ocular melanoma; vitronectin; fibrinogen neurologicaltumors α_(V)β₃ vitronectin activated endothelial cells, vitronectin,fibronectin, fibrinogen, receptor melanoma, glioblastoma osteopontin,Cyr61, thyroxine, TETRAC α_(V)β₅ widespread, esp. fibroblasts,vitronectin and adenovirus epithelial cells α_(V)β₆ proliferatingepithelia, esp. fibronectin; T6Eβ1 + 3 lung and mammary gland α_(V)β₈neural tissue; peripheral fibronectin; T6Eβ1 + 3 nerve α₆β₄ Epithelialcells Laminin

Thus, in certain embodiments, the at least one NRP2 ligand is selectedfrom Table N2 and/or Table N3.

For example, in some aspects, the at least one NRP2 ligand is a VEGF(vascular endothelial growth factor) ligand selected from VEGF-A145,VEGF-A165, VEGF-C, VEGF-D, and PIGF-2. VEGF-VEGFR2/3-NRP2 interactionsare associated with promoting cell migration, cell growth, cellsurvival, and cell attachment, and also with lymphangiogenesis,increasing vascular permeability, activating integrin signaling,promoting vesicular trafficking and internalization, and slowingcellular differentiation. Accordingly, anti-NRP2 antibodies whichmodulate VEGF related NRP2 ligands would be expected find utility inmodulating one or more of these pathways.

In certain aspects, the at least one NRP2 ligand is a semaphorinselected from one or more of SEMA-3B, SEMA-3C, SEMA-3D, SEMA-3F, andSEMA-3B, or a plexin receptor selected from one or more of plexins A1,A2, A3, A4, and D1. SEMAs typically antagonize the effects of VEGF-C,through there is a close dynamic interplay between VEGF and Semasignaling pathways. SEMAs typically function in the immune system tocontrol cell movement, cell migration, cell-cell communication, and cellactivation. SEMA Plexin-NRP2 interactions are associated with inhibitingcell migration, inhibiting cell growth, promoting apoptosis, inhibitingcell attachment, inhibiting integrin signaling, promoting cellulardifferentiation, inhibiting lymphangiogenesis, reducing vascularpermeability, promoting microtubule destabilization, mediating thecollapse of actin cytoskeleton & cell contraction including growth conecollapse and actomyosin contraction, and preventing neuronal cellspreading and inhibiting axon outgrowth. Accordingly, anti-NRP2antibodies which modulate SEMA-related NRP2 ligands would be expectedfind utility in modulating one or more of these pathways.

In some aspects, the at least one NRP2 ligand is an integrin selectedfrom one or more of αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α6β1 and α64β.Integrin-NRP2 interactions are generally associated with increased celladhesion, cell growth, cancer growth and invasiveness. Accordinglyanti-NRP2 antibodies which modulate integrin related NRP2 ligands wouldbe expected find utility in modulating one or more of these pathways.

In some aspects, the at least one NRP2 ligand is selected from TGFβ1,TGFβ2, TGFβ3, and their corresponding TGFβ receptors. TGF-β signaling isstrongly involved in the regulation of EMT in cancer, and also infibrosis development (see, for example, Gemmill et al., Sci. Signal. 10eaag0528, 2017). NRP2b expression is preferentially upregulated byβTGF-@ signaling in abnormal lungs, and shows little or no expression innormal lung. NRP2b expression enhances migration, invasion, metastasis,chemoresistance, and tumorsphere formation, and also enhances acquiredEGFR inhibitor resistance associated with EMT in cancer cells.Accordingly, anti-NRP2 antibodies which modulate TGF-@ related NRP2ligands would be expected find utility in modulating one or more ofthese pathways, and find utility in the treatment of cancerchemoresistance. In certain embodiments, an anti-NRP2 antibody orantigen-binding fragment thereof modulates binding/signaling activitybetween an NRP2 polypeptide and at least one of the NRP2 ligands fromTable N2 and/or Table N3, for example, by specifically binding to anNRP2 ligand-interacting region of the NRP2 polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof binds selectively to the NRP2a isoform (e.g., variants1, 2, and/or 3 of Table N1) of NRP2, and does not substantially bind tothe NRP2b isoform (e.g., variants 4 and/or 5 of Table N1) of NRP2. Insome embodiments, the at least one antibody or antigen-binding fragmentthereof binds selectively to the NRP2b isoform (e.g., variants 4 and/or5 of Table N1), and does not substantially bind to the NRP2a isoform(e.g., variants 1, 2, and/or 3 of Table N1) of NRP2.

In some instances, the at least one antibody or antigen-binding fragmentthereof antagonizes the binding/signaling activity between the NRP2polypeptide and the at least one NRP2 ligand. For example, in someembodiments, the anti-NRP2 antibody antagonizes or reduces thetheoretical maximal binding/signaling between the NRP2 polypeptide andthe NRP2 ligand by about or at least about 20-100% (e.g., about 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, or 100%) afterpre-incubation of the anti-NRP2 antibody with the NRP2polypeptide/ligand in a substantially stoichiometrically equivalentamount.

In some instances, the at least one antibody or antigen-binding fragmentthereof reduces or inhibits the dimerization between two NRP2polypeptides. For example, in some embodiments, the anti-NRP2 antibodyantagonizes or reduces the theoretical maximal dimerization between twoNRP2 polypeptides by about or at least about 20-100% (e.g., about 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, or 100%) afterpre-incubation of the anti-NRP2 antibody with the NRP2 polypeptides in asubstantially stoichiometrically equivalent amount.

In some instances, the at least one antibody or antigen-binding fragmentthereof agonizes or enhances the dimerization between two NRP2polypeptides. For instance, in some embodiments, the anti-NRP2 antibodyagonizes or enhances the basal dimerization state of two NRP2polypeptides by about or at least about 20%-500% (e.g., about 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500%) after pre-incubationof the anti-NRP2 antibody with the NRP2 polypeptides in a substantiallystoichiometrically equivalent amount.

In some instances, the at least one antibody or antigen-binding fragmentthereof agonizes or enhances the binding/signaling activity between theNRP2 polypeptide and the at least one NRP2 ligand. For instance, in someembodiments, the anti-NRP2 antibody agonizes or enhances the theoreticalmaximal binding/signaling activity between the NRP2 polypeptide and theat least one NRP2 ligand by about or at least about 20%-500% (e.g.,about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500%) afterpre-incubation of the anti-NRP2 antibody with the NRP2 polypeptide in asubstantially stoichiometrically equivalent amount.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof selectively modulates the binding and/or signaling ofsemaphorins to, or via, the NRP2 polypeptide. In some aspects, suchantibodies do not substantially block the interaction of VEGF-C orrelated NRP2 ligands. In some aspects, such antibodies are agonisticantibodies with respect to semaphorin signaling. In some aspects, suchantibodies are antagonistic antibodies with respect to semaphorinsignaling.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof selectively modulates the binding and/or signaling ofVEGF-C or related NRP2 ligands to, or via, the NRP2 polypeptide. In someaspects, such antibodies do not substantially block the interaction ofsemaphorins. In some embodiments, such antibodies selectively modulateboth the binding of VEGF-C or related NRP2 ligands and semaphorins tothe NRP2 polypeptide. In some embodiments, such antibodies are agonisticantibodies with respect to VEGF-C signaling. In some aspects, suchantibodies are antagonistic antibodies with respect to VEGF-C signaling.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof selectively modulates the binding and/or signaling ofintegrins or related NRP2 ligands to the NRP2 polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof selectively modulates the binding and/or signaling ofTGFβ1, TGFβ2, TGFβ3, or their corresponding TGFβ receptors to the NRP2polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof selectively modulates the binding and/or signaling offibroblast growth factor (FGF), galectin, hepatocyte growth factor(HGF), platelet derived growth factor, and/or their correspondingreceptors to the NRP2 polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor and/or a semaphorin withoutsubstantially modulating the binding/signaling activity between the NRP2polypeptide and VEGFR2, VEGFR3, and/or VEGF-C.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor and/or semaphorin withoutsubstantially modulating the binding/signaling activity between the NRP2polypeptide and a HRS polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor and/or a semaphorin withoutsubstantially modulating the binding/signaling activity between the NRP2polypeptide and a HRS polypeptide, and without substantially modulatingthe binding/signaling activity between the NRP2 polypeptide and VEGFR2,VEGFR3, and/or VEGF-C.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and VEGFR2 and/or VEGFR3 without substantiallymodulating the binding/signaling activity between the NRP2 polypeptideand a plexin receptor and/or a semaphorin.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and VEGFR2, VEGFR3, and/or VEGF-C without substantiallymodulating the binding/signaling activity between the NRP2 polypeptideand a HRS polypeptide.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between theNRP2 polypeptide and a plexin receptor without substantially modulatingthe ligand binding of semaphorin 3 to NRP2.

In some embodiments, the plexin receptor is selected from plexin A1, A2,A3, A4, and D1. In some embodiments, the semaphorin is selected fromsemaphorin 3B, 3C, 3D, 3F, and 3G.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope of at least 5 aminoacids within the human NRP2 a2 domain, wherein the at least one antibodyor antigen-binding fragment thereof selectively inhibits receptordimerization between NRP2 and plexin A1 without substantially inhibitingdimerization between NRP2 and FLT4 (VEGFR3). In some embodiments, the atleast one antibody or antigen-binding fragment thereof specificallybinds to an epitope within amino acids 232-242 of a human NRP2 precursor(see Table N1).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope of at least 5 aminoacids within the human NRP2 b1 domain, wherein the at least one antibodyor antigen-binding fragment thereof selectively inhibits receptordimerization between NRP2 and FLT4 (VEGFR3) without substantiallyinhibiting dimerization between NRP2 and plexin A1.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope of at least 5 aminoacids within the human NRP2 b2 domain, wherein the at least one antibodyor antigen-binding fragment thereof inhibits receptor dimerizationbetween NRP2 and FLT4 (VEGFR3) and inhibits dimerization between NRP2and plexin A1.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof specifically binds to an epitope of at least 5 aminoacids within the human NRP2 c domain, wherein the at least one antibodyor antigen-binding fragment thereof inhibits receptor dimerizationbetween NRP2 and plexin A1 and partially inhibits dimerization betweenNRP2 and FLT4 (VEGFR3).

In some embodiments, the at least one antibody or antigen-bindingfragment thereof has an affinity (Kd or EC₅₀) for each of (i) a humanNRP2 polypeptide and (ii) the corresponding region of a cynomolgusmonkey NRP2 polypeptide (see, for example, UniProt G7PL91), wherein theaffinity for (i) and (ii) is within the range of about 20 pM to about200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about100 pM to about 1 nM, about 0.4 to about 1.2 nM, about 0.9 to about 5.5nM, about 0.9 to about 5 nM, or about 1 nM to about 10 nM.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof has an affinity (Kd or EC₅₀) for each of (i) a humanNRP2 polypeptide and (ii) the corresponding region of a murine NRP2polypeptide, wherein the affinity for (i) and (ii) is within the rangeof about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pMto about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90pM to about 900 pM, about 100 pM to about 1 nM, or about 1 nM to about10 nM.

In certain embodiments, the at least one antibody or antigen-bindingfragment thereof binds selectively to a human NRP2 polypeptide (seeTable N1) relative to a murine NRP2 polypeptide, for instance, where itsaffinity for a human NRP2 polypeptide is significantly stronger than itsaffinity for a murine NRP2 polypeptide, for example, by about or atleast about 2, 5, 10, 20, 30, 40, 50, 100, 500, or 1000-fold or more. Inparticular embodiments, the at least one antibody or antigen-bindingfragment thereof binds selectively to a human NRP2 polypeptide and doesnot substantially bind to a murine NRP2 polypeptide. Certain exemplarymurine NRP2 polypeptides include the Mus musculus NRP2 polypeptide (see,for example, UniProt 035375).

Merely for illustrative purposes, the binding interactions between ahuman NRP2 polypeptide and an NRP2 ligand can be detected and quantifiedusing a variety of routine methods, including biacore assays (forexample, with appropriately tagged soluble reagents, bound to a sensorchip), FACS analyses with cells expressing a NRP2 polypeptide on thecell surface (either native, or recombinant), immunoassays, fluorescencestaining assays, ELISA assays, and microcalorimetry approaches such asITC (Isothermal Titration Calorimetry).

In certain embodiments, an antibody or antigen-binding fragment thereofcomprises variant or otherwise modified Fc region(s), including thosehaving altered properties or biological activities relative to wild-typeFc region(s). Examples of modified Fc regions include those havingmutated sequences, for instance, by substitution, insertion, deletion,or truncation of one or more amino acids relative to a wild-typesequence, hybrid Fc polypeptides composed of domains from differentimmunoglobulin classes/subclasses, Fc polypeptides having alteredglycosylation/sialylation patterns, and Fc polypeptides that aremodified or derivatized, for example, by biotinylation (see, e.g., USApplication No. 2010/0209424), phosphorylation, sulfation, etc., or anycombination of the foregoing. Such modifications can be employed toalter (e.g., increase, decrease) the binding properties of the Fc regionto one or more particular FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc,FcγRIIIa, FcγRIIIb, FcRn), its pharmacokinetic properties (e.g.,stability or half-life, bioavailability, tissue distribution, volume ofdistribution, concentration, elimination rate constant, eliminationrate, area under the curve (AUC), clearance, C_(max), t_(max), C_(min),fluctuation), its immunogenicity, its complement fixation or activation,and/or the CDC/ADCC/ADCP-related activities of the Fc region, amongother properties described herein, relative to a corresponding wild-typeFc sequence of an antibody or antigen-binding fragment thereof. Includedare modified Fc regions of human and/or mouse origin.

Also included are antibodies or antigen-binding fragments thereof thatcomprise hybrid Fc regions, for example, Fc regions that comprise acombination of Fc domains (e.g., hinge, CH₂, CH₃, CH₄) fromimmunoglobulins of different species (e.g., human, mouse), different Igclasses, and/or different Ig subclasses. General examples include hybridFc regions that comprise, consist of, or consist essentially of thefollowing combination of CH₂/CH₃ domains: IgA1/IgA1, IgA1/IgA2,IgA1/IgD, IgA1/IgE, IgA1/IgG1, IgA1/IgG2, IgA1/IgG3, IgA1/IgG4,IgA1/IgM, IgA2/IgA1, IgA2/IgA2, IgA2/IgD, IgA2/IgE, IgA2/IgG1,IgA2/IgG2, IgA2/IgG3, IgA2/IgG4, IgA2/IgM, IgD/IgA1, IgD/IgA2, IgD/IgD,IgD/IgE, IgD/IgG1, IgD/IgG2, IgD/IgG3, IgD/IgG4, IgD/IgM, IgE/IgA1,IgE/IgA2, IgE/IgD, IgE/IgE, IgE/IgG1, IgE/IgG2, IgE/IgG3, IgE/IgG4,IgE/IgM, IgG1/IgA1, IgG1/IgA2, IgG1/IgD, IgG1/IgE, IgG1/IgG1, IgG1/IgG2,IgG1/IgG3, IgG1/IgG4, IgG1/IgM, IgG2/IgA1, IgG2/IgA2, IgG2/IgD,IgG2/IgE, IgG2/IgG1, IgG2/IgG2, IgG2/IgG3, IgG2/IgG4, IgG2/IgM,IgG3/IgA1, IgG3/IgA2, IgG3/IgD, IgG3/IgE, IgG3/IgG1, IgG3/IgG2,IgG3/IgG3, IgG3/IgG4, IgG3/IgM, IgG4/IgA1, IgG4/IgA2, IgG4/IgD,IgG4/IgE, IgG4/IgG1, IgG4/IgG2, IgG4/IgG3, IgG4/IgG4, IgG4/IgM,IgM/IgA1, IgM/IgA2, IgM/IgD, IgM/IgE, IgM/IgG1, IgM/IgG2, IgM/IgG3,IgM/IgG4, IgM/IgM (or fragments or variants thereof), and optionallyinclude a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3,or IgG4, and/or a CH₄ domain from IgE and/or IgM. In specificembodiments, the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Additional examples include hybrid Fc regions that comprise, consist of,or consist essentially of the following combination of CH₂/CH₄ domains:IgA1/IgE, IgA2/IgE, IgD/IgE, IgE/IgE, IgG1/IgE, IgG2/IgE, IgG3/IgE,IgG4/IgE, IgM/IgE, IgA1/IgM, IgA2/IgM, IgD/IgM, IgE/IgM, IgG1/IgM,IgG2/IgM, IgG3/IgM, IgG4/IgM, IgM/IgM (or fragments or variantsthereof), and optionally include a hinge from one or more of IgA1, IgA2,IgD, IgG1, IgG2, IgG3, IgG4, and/or a CH₃ domain from one or more ofIgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In specificembodiments, the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Certain examples include hybrid Fc regions that comprise, consist of, orconsist essentially of the following combination of CH₃/CH₄ domains:IgA1/IgE, IgA2/IgE, IgD/IgE, IgE/IgE, IgG1/IgE, IgG2/IgE, IgG3/IgE,IgG4/IgE, IgM/IgE, IgA1/IgM, IgA2/IgM, IgD/IgM, IgE/IgM, IgG1/IgM,IgG2/IgM, IgG3/IgM, IgG4/IgM, IgM/IgM (or fragments or variantsthereof), and optionally include a hinge from one or more of IgA1, IgA2,IgD, IgG1, IgG2, IgG3, IgG4, and/or a CH₂ domain from one or more ofIgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In specificembodiments, the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Particular examples include hybrid Fc regions that comprise, consist of,or consist essentially of the following combination of hinge/CH₂domains: IgA1/IgA1, IgA1/IgA2, IgA1/IgD, IgA1/IgE, IgA1/IgG1, IgA1/IgG2,IgA1/IgG3, IgA1/IgG4, IgA1/IgM, IgA2/IgA1, IgA2/IgA2, IgA2/IgD,IgA2/IgE, IgA2/IgG1, IgA2/IgG2, IgA2/IgG3, IgA2/IgG4, IgA2/IgM,IgD/IgA1, IgD/IgA2, IgD/IgD, IgD/IgE, IgD/IgG1, IgD/IgG2, IgD/IgG3,IgD/IgG4, IgD/IgM, IgG1/IgA1, IgG1/IgA2, IgG1/IgD, IgG1/IgE, IgG1/IgG1,IgG1/IgG2, IgG1/IgG3, IgG1/IgG4, IgG1/IgM, IgG2/IgA1, IgG2/IgA2,IgG2/IgD, IgG2/IgE, IgG2/IgG1, IgG2/IgG2, IgG2/IgG3, IgG2/IgG4,IgG2/IgM, IgG3/IgA1, IgG3/IgA2, IgG3/IgD, IgG3/IgE, IgG3/IgG1,IgG3/IgG2, IgG3/IgG3, IgG3/IgG4, IgG3/IgM, IgG4/IgA1, IgG4/IgA2,IgG4/IgD, IgG4/IgE, IgG4/IgG1, IgG4/IgG2, IgG4/IgG3, IgG4/IgG4, IgG4/IgM(or fragments or variants thereof), and optionally include a CH₃ domainfrom one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, orIgM, and/or a CH₄ domain from IgE and/or IgM. In specific embodiments,the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Certain examples include hybrid Fc regions that comprise, consist of, orconsist essentially of the following combination of hinge/CH₃ domains:IgA1/IgA1, IgA1/IgA2, IgA1/IgD, IgA1/IgE, IgA1/IgG1, IgA1/IgG2,IgA1/IgG3, IgA1/IgG4, IgA1/IgM, IgA2/IgA1, IgA2/IgA2, IgA2/IgD,IgA2/IgE, IgA2/IgG1, IgA2/IgG2, IgA2/IgG3, IgA2/IgG4, IgA2/IgM,IgD/IgA1, IgD/IgA2, IgD/IgD, IgD/IgE, IgD/IgG1, IgD/IgG2, IgD/IgG3,IgD/IgG4, IgD/IgM, IgG1/IgA1, IgG1/IgA2, IgG1/IgD, IgG1/IgE, IgG1/IgG1,IgG1/IgG2, IgG1/IgG3, IgG1/IgG4, IgG1/IgM, IgG2/IgA1, IgG2/IgA2,IgG2/IgD, IgG2/IgE, IgG2/IgG1, IgG2/IgG2, IgG2/IgG3, IgG2/IgG4,IgG2/IgM, IgG3/IgA1, IgG3/IgA2, IgG3/IgD, IgG3/IgE, IgG3/IgG1,IgG3/IgG2, IgG3/IgG3, IgG3/IgG4, IgG3/IgM, IgG4/IgA1, IgG4/IgA2,IgG4/IgD, IgG4/IgE, IgG4/IgG1, IgG4/IgG2, IgG4/IgG3, IgG4/IgG4, IgG4/IgM(or fragments or variants thereof), and optionally include a CH₂ domainfrom one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, orIgM, and/or a CH₄ domain from IgE and/or IgM. In specific embodiments,the hinge, CH₂, CH₃, and CH₄ domains are from human Ig.

Some examples include hybrid Fc regions that comprise, consist of, orconsist essentially of the following combination of hinge/CH₄ domains:IgA1/IgE, IgA1/IgM, IgA2/IgE, IgA2/IgM, IgD/IgE, IgD/IgM, IgG1/IgE,IgG1/IgM, IgG2/IgE, IgG2/IgM, IgG3/IgE, IgG3/IgM, IgG4/IgE, IgG4/IgM (orfragments or variants thereof), and optionally include a CH₂ domain fromone or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM,and/or a CH₃ domain from one or more of IgA1, IgA2, IgD, IgE, IgG1,IgG2, IgG3, IgG4, or IgM.

Specific examples of hybrid Fc regions can be found, for example, in WO2008/147143, which are derived from combinations of IgG subclasses orcombinations of human IgD and IgG.

Also included are antibodies or antigen-binding fragments thereof havingderivatized or otherwise modified Fc regions. In certain aspects, the Fcregion may be modified by phosphorylation, sulfation, acrylation,glycosylation, methylation, farnesylation, acetylation, amidation, andthe like, for instance, relative to a wild-type or naturally-occurringFc region. In certain embodiments, the Fc region may comprise wild-typeor native glycosylation patterns, or alternatively, it may compriseincreased glycosylation relative to a native form, decreasedglycosylation relative to a native form, or it may be entirelydeglycosylated. As one example of a modified Fc glycoform, decreasedglycosylation of an Fc region reduces binding to the C1q region of thefirst complement component C1, a decrease in ADCC-related activity,and/or a decrease in CDC-related activity. Certain embodiments thusemploy a deglycosylated or aglycosylated Fc region. See, e.g., WO2005/047337 for the production of exemplary aglycosylated Fc regions.Another example of an Fc region glycoform can be generated bysubstituting the Q295 position with a cysteine residue (see, e.g., U.S.Application No. 2010/0080794), according to the Kabat et al. numberingsystem. Certain embodiments may include Fc regions where about 80-100%of the glycoprotein in Fc region comprises a mature core carbohydratestructure that lacks fructose (see, e.g., U.S. Application No.2010/0255013). Some embodiments may include Fc regions that areoptimized by substitution or deletion to reduce the level offucosylation, for instance, to increase affinity for FcγRI, FcγRIa, orFcγRIIIa, and/or to improve phagocytosis by FcγRIIa-expressing cells(see U.S. Application Nos. 2010/0249382 and 2007/0148170).

As another example of a modified Fc glycoform, an Fc region of anantibody or antigen-binding fragment thereof may compriseoligomannose-type N-glycans, and optionally have one or more of thefollowing: increased ADCC effector activity, increased binding affinityfor FcγRIIIA (and certain other FcRs), similar or increased bindingspecificity for the target of the NRP2 polypeptide, similar or higherbinding affinity for the target of the NRP2 polypeptide, and/or similaror lower binding affinity for mannose receptor, relative to acorresponding Fc region that contains complex-type N-glycans (see, e.g.,U.S. Application No. 2007/0092521 and U.S. Pat. No. 7,700,321). Asanother example, enhanced affinity of Fc regions for FcγRs has beenachieved using engineered glycoforms generated by expression ofantibodies in engineered or variant cell lines (see, e.g., Umana et al.,Nat Biotechnol. 17:176-180, 1999; Davies et al., Biotechnol Bioeng.74:288-294, 2001; Shields et al., J Biol Chem. 277:26733-26740, 2002;Shinkawa et al., 2003, J Biol Chem. 278:3466-3473, 2003; and U.S.Application No. 2007/0111281). Certain Fc region glycoforms comprise anincreased proportion of N-glycoside bond type complex sugar chains,which do not have the 1-position of fucose bound to the 6-position ofN-acetylglucosamine at the reducing end of the sugar chain (see, e.g.,U.S. Application No. 2010/0092997). Particular embodiments may includeIgG Fc region that is glycosylated with at least one galactose moietyconnected to a respective terminal sialic acid moiety by an α-2,6linkage, optionally where the Fc region has a higher anti-inflammatoryactivity relative to a corresponding, wild-type Fc region (see U.S.Application No. 2008/0206246). Certain of these and related alteredglycosylation approaches have generated substantial enhancements of thecapacity of Fc regions to selectively bind FcRs such as FcγRIII, tomediate ADCC, and to alter other properties of Fc regions, as describedherein.

Certain variant, fragment, hybrid, or otherwise modified Fc regions ofan antibody or antigen-binding fragment thereof may have altered bindingto one or more FcRs, and/or corresponding changes to effector function,relative to a corresponding, wild-type Fc sequence (e.g., same species,same Ig class, same Ig subclass). For instance, such Fc regions may haveincreased binding to one or more of Fcγ receptors, Fcα receptors, Fcεreceptors, and/or the neonatal Fc receptor, relative to a corresponding,wild-type Fc sequence. In other embodiments, variant, fragment, hybrid,or modified Fc regions may have decreased binding to one or more of Fcγreceptors, Fcα receptors, Fcε receptors, and/or the neonatal Fcreceptor, relative to a corresponding, wild-type Fc sequence. SpecificFcRs are described elsewhere herein.

In some embodiments, an antibody comprises an Fc domain, comprising oneor more mutations to increase binding to one or more of Fcγ receptors,Fcα receptors, Fcε receptors, and/or the neonatal Fc receptor, relativeto a corresponding, wild-type Fc sequence. In some embodiments, anantibody comprises an IgG1 or IgG3 Fc domain, comprising one or moremutations to increase binding to one or more of Fcγ receptors, Fcαreceptors, Fcε receptors, and/or the neonatal Fc receptor, relative to acorresponding, wild-type Fc sequence. In some embodiments, an antibodycomprises an Fc domain, comprising one or more mutations to increaseeffector function. In some embodiments the at least one antibodycomprises an Fc domain selected from a human IgG1 and IgG3, comprisingone or more mutations to increase effector function.

In some embodiments, an antibody is blocking antibody that comprises anFc domain with high effector activity. In some embodiments, the blockingantibody comprises an Fc domain selected from a human IgG1 and IgG3,comprising one or more mutations to increase effector function. In someembodiments, an antibody is a partial-blocking antibody that comprisesan Fc domain with high effector activity. In some embodiments, the apartial-blocking antibody comprises an Fc domain selected from a humanIgG1 and IgG3, comprising one or more mutations to increase effectorfunction. In some embodiments, an antibody is a non-blocking antibodythat comprises an Fc domain with high effector activity. In someembodiments, the non-blocking antibody comprises an Fc domain selectedfrom a human IgG1 or IgG3, comprising one or more mutations to increaseeffector function.

In some embodiments, an antibody comprises an Fc domain, comprising oneor more mutations to decrease binding to one or more of Fcγ receptors,Fcα receptors, Fcε receptors, and/or the neonatal Fc receptor, relativeto a corresponding, wild-type Fc sequence. In some embodiments, anantibody comprises an IgG1 or IgG3 Fc domain, comprising one or moremutations to decrease binding to one or more of Fcγ receptors, Fcαreceptors, Fcε receptors, and/or the neonatal Fc receptor, relative to acorresponding, wild-type Fc sequence. In some embodiments, an antibodycomprises an Fc domain, comprising one or more mutations to decreaseeffector function. In some embodiments, an antibody comprises an Fcdomain selected from a human IgG2 and IgG4, comprising one or moremutations to decrease effector function.

In some embodiments, an antibody is a blocking antibody comprising an Fcdomain with low effector activity. In some embodiments, the blockingantibody comprises an Fc domain selected from a human IgG2 and IgG4,comprising one or more mutations to decrease effector function. In someembodiments, an antibody is a partial-blocking antibody comprising an Fcdomain with low effector activity. In some embodiments, thepartial-blocking antibody comprises an Fc domain selected from a humanIgG2 and IgG4, comprising one or more mutations to decrease effectorfunction. In some embodiments, an antibody is a non-blocking antibodycomprising an Fc domain with low effector activity. In some embodiments,the non-blocking antibody comprises an Fc domain selected from a humanIgG2 and IgG4, comprising one or more mutations to decrease effectorfunction.

Specific examples of Fc variants having altered (e.g., increased,decreased) effector function/FcR binding can be found, for example, inU.S. Pat. Nos. 5,624,821 and 7,425,619; U.S. Application Nos.2009/0017023, 2009/0010921, and 2010/0203046; and WO 2000/42072 and WO2004/016750. Certain examples include human Fc regions having a one ormore substitutions at position 298, 333, and/or 334, for example, S298A,E333A, and/or K334A (based on the numbering of the EU index of Kabat etal.), which have been shown to increase binding to the activatingreceptor FcγRIIIa and reduce binding to the inhibitory receptor FcγRIIb.These mutations can be combined to obtain double and triple mutationvariants that have further improvements in binding to FcRs. Certainembodiments include a S298A/E333A/K334A triple mutant, which hasincreased binding to FcγRIIIa, decreased binding to FcγRIIb, andincreased ADCC (see, e.g., Shields et al., J Biol Chem. 276:6591-6604,2001; and Presta et al., Biochem Soc Trans. 30:487-490, 2002). See alsoengineered Fc glycoforms that have increased binding to FcRs, asdisclosed in Umana et al., supra; and U.S. Pat. No. 7,662,925. Someembodiments include Fc regions that comprise one or more substitutionsselected from 434S, 252Y/428L, 252Y/434S, and 428L/434S (see U.S.Application Nos. 2009/0163699 and 20060173170), based on the EU index ofKabat et al.

Certain variant, fragment, hybrid, or modified Fc regions may havealtered effector functions, relative to a corresponding, wild-type Fcsequence. For example, such Fc regions may have increased complementfixation or activation, increased Clq binding affinity, increasedCDC-related activity, increased ADCC-related activity, and/or increasedADCP-related activity, relative to a corresponding, wild-type Fcsequence. In other embodiments, such Fc regions may have decreasedcomplement fixation or activation, decreased Clq binding affinity,decreased CDC-related activity, decreased ADCC-related activity, and/ordecreased ADCP-related activity, relative to a corresponding, wild-typeFc sequence. As merely one illustrative example, an Fc region maycomprise a deletion or substitution in a complement-binding site, suchas a C1q-binding site, and/or a deletion or substitution in an ADCCsite. Examples of such deletions/substitutions are described, forexample, in U.S. Pat. No. 7,030,226. Many Fc effector functions, such asADCC, can be assayed according to routine techniques in the art. (see,e.g., Zuckerman et al., CRC Crit Rev Microbiol. 7:1-26, 1978). Usefuleffector cells for such assays includes, but are not limited to, naturalkiller (NK) cells, macrophages, and other peripheral blood mononuclearcells (PBMC). Alternatively, or additionally, certain Fc effectorfunctions may be assessed in vivo, for example, by employing an animalmodel described in Clynes et al. PNAS. 95:652-656, 1998.

Certain variant hybrid, or modified Fc regions may have alteredstability or half-life relative to a corresponding, wild-type Fcsequence. In certain embodiments, such Fc regions may have increasedhalf-life relative to a corresponding, wild-type Fc sequence. In otherembodiments, variant hybrid, or modified Fc regions may have decreasedhalf-life relative to a corresponding, wild-type Fc sequence. Half-lifecan be measured in vitro (e.g., under physiological conditions) or invivo, according to routine techniques in the art, such as radiolabeling,ELISA, or other methods. In vivo measurements of stability or half-lifecan be measured in one or more bodily fluids, including blood, serum,plasma, urine, or cerebrospinal fluid, or a given tissue, such as theliver, kidneys, muscle, central nervous system tissues, bone, etc.

As one example, modifications to an Fc region that alter its ability tobind the FcRn can alter its half-life in vivo, or other properties. Insome embodiments, a modified Fc domain, for example, a modified IgG1 orIgG4 Fc domain, comprises at least one mutation to alter FcRn binding asdescribed, for example, by Zalevsky et al. (Nature Biotechnology. 28(2):157-159, 2010) or Mackness et al. (mAbs. 11(7): 1276-1288, 2019). Inspecific embodiments, a modified IgG1 or IgG3 Fc domain comprises anyone or more of YD (M252Y/T256D), DQ (T256D/T307Q), DW (T256D/T307W), YTE(M252Y/S254T/T256E), AAA (T307A/E380A/N434A), LS (M428L/N434S), M252Y,T256D/E, K288D/N, T307Q/W, E380C, N434FY, or Y436H/N/W mutations (EUnumbering), including combinations thereof. In some embodiments, amodified IgG1 or IgG3 Fc domain comprises any one or more of M252Y,T256D/E, T307Q/W, and/or N434F/Y mutations (EU numbering), includingcombinations thereof. Assays for measuring the in vivo pharmacokineticproperties (e.g., in vivo mean elimination half-life) and non-limitingexamples of Fc modifications that alter its binding to the FcRn aredescribed, for example, in U.S. Pat. Nos. 7,217,797 and 7,732,570; andU.S. Application Nos. US 2010/0143254 and 2010/0143254.

Additional non-limiting examples of modifications to alter stability orhalf-life include substitutions/deletions at one or more of amino acidresidues selected from 251-256, 285-290, and 308-314 in the CH₂ domain,and 385-389 and 428-436 in the CH₃ domain, according to the numberingsystem of Kabat et al. See U.S. Application No. 2003/0190311. Specificexamples include substitution with leucine at position 251, substitutionwith tyrosine, tryptophan or phenylalanine at position 252, substitutionwith threonine or serine at position 254, substitution with arginine atposition 255, substitution with glutamine, arginine, serine, threonine,or glutamate at position 256, substitution with threonine at position308, substitution with proline at position 309, substitution with serineat position 311, substitution with aspartate at position 312,substitution with leucine at position 314, substitution with arginine,aspartate or serine at position 385, substitution with threonine orproline at position 386, substitution with arginine or proline atposition 387, substitution with proline, asparagine or serine atposition 389, substitution with methionine or threonine at position 428,substitution with tyrosine or phenylalanine at position 434,substitution with histidine, arginine, lysine or serine at position 433,and/or substitution with histidine, tyrosine, arginine or threonine atposition 436, including any combination thereof. Such modificationsoptionally increase affinity of the Fc region for the FcRn and therebyincrease half-life, relative to a corresponding, wild-type Fc region.

Certain variant hybrid, or modified Fc regions may have alteredsolubility relative to a corresponding, wild-type Fc sequence. Incertain embodiments, such Fc regions may have increased solubilityrelative to a corresponding, wild-type Fc sequence. In otherembodiments, variant hybrid, or modified Fc regions may have decreasedsolubility relative to a corresponding, wild-type Fc sequence.Solubility can be measured, for example, in vitro (e.g., underphysiological conditions) according to routine techniques in the art.Exemplary solubility measurements are described elsewhere herein.

Additional examples of variants include IgG Fc regions havingconservative or non-conservative substitutions (as described elsewhereherein) at one or more of positions 250, 314, or 428 of the heavy chain,or in any combination thereof, such as at positions 250 and 428, or atpositions 250 and 314, or at positions 314 and 428, or at positions 250,314, and 428 (see, e.g., U.S. Application No. 2011/0183412). In specificembodiments, the residue at position 250 is substituted with glutamicacid or glutamine, and/or the residue at position 428 is substitutedwith leucine or phenylalanine. As another illustrative example of an IgGFc variant, any one or more of the amino acid residues at positions 214to 238, 297 to 299, 318 to 322, and/or 327 to 331 may be used as asuitable target for modification (e.g., conservative or non-conservativesubstitution, deletion). In particular embodiments, the IgG Fc variantCH₂ domain contains amino acid substitutions at positions 228, 234, 235,and/or 331 (e.g., human IgG4 with Ser228Pro and Leu235Ala mutations) toattenuate the effector functions of the Fc region (see U.S. Pat. No.7,030,226). Here, the numbering of the residues in the heavy chain isthat of the EU index (see Kabat et al., “Sequences of Proteins ofImmunological Interest,” 5^(th) Ed., National Institutes of Health,Bethesda, Md. (1991)). Certain of these and related embodiments havealtered (e.g., increased, decreased) FcRn binding and/or serumhalf-life, optionally without reduced effector functions such as ADCC orCDC-related activities.

Additional examples include variant Fc regions that comprise one or moreamino acid substitutions at positions 279, 341, 343 or 373 of awild-type Fc region, or any combination thereof (see, e.g., U.S.Application No. 2007/0224188). The wild-type amino acid residues atthese positions for human IgG are valine (279), glycine (341), proline(343) and tyrosine (373). The substation(s) can be conservative ornon-conservative, or can include non-naturally occurring amino acids ormimetics, as described herein. Alone or in combination with thesesubstitutions, certain embodiments may also employ a variant Fc regionthat comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsubstitutions selected from the following: 235G, 235R, 236F, 236R, 236Y,237K, 237N, 237R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 244L,245R, 247A, 247D, 247E, 247F, 247M, 247N, 247Q, 247R, 247S, 247T, 247W,247Y, 248F, 248P, 248Q, 248W, 249L, 249M, 249N, 249P, 249Y, 251H, 251I,251W, 254D, 254E, 254F, 254G, 254H, 254I, 254K, 254L, 254M, 254N, 254P,254Q, 254R, 254V, 254W, 254Y, 255K, 255N, 256H, 256I, 256K, 256L, 256V,256W, 256Y, 257A, 257I, 257M, 257N, 257S, 258D, 260S, 262L, 264S, 265K,265S, 267H, 267I, 267K, 268K, 269N, 269Q, 271T, 272H, 272K, 272L, 272R,279A, 279D, 279F, 279G, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R,279S, 279T, 279W, 279Y, 280T, 283F, 283G, 283H, 283I, 283K, 283L, 283M,283P, 283R, 283T, 283W, 283Y, 285N, 286F, 288N, 288P, 292E, 292F, 292G,292I, 292L, 293S, 293V, 301W, 304E, 307E, 307M, 312P, 315F, 315K, 315L,315P, 315R, 316F, 316K, 317P, 317T, 318N, 318P, 318T, 332F, 332G, 332L,332M, 332S, 332V, 332W, 339D, 339E, 339F, 339G, 339H, 339I, 339K, 339L,339M, 339N, 339Q, 339R, 339S, 339W, 339Y, 341D, 341E, 341F, 341H, 341I,341K, 341L, 341M, 341N, 341P, 341Q, 341R, 341S, 341T, 341V, 341W, 341Y,343A, 343D, 343E, 343F, 343G, 343H, 343I, 343K, 343L, 343M, 343N, 343Q,343R, 343S, 343T, 343V, 343W, 343Y, 373D, 373E, 373F, 373G, 373H, 373I,373K, 373L, 373M, 373N, 373Q, 373R, 373S, 373T, 373V, 373W, 375R, 376E,376F, 376G, 376H, 376I, 376L, 376M, 376N, 376P, 376Q, 376R, 376S, 376T,376V, 376W, 376Y, 377G, 377K, 377P, 378N, 379N, 379Q, 379S, 379T, 380D,380N, 380S, 380T, 382D, 382F, 382H, 382I, 382K, 382L, 382M, 382N, 382P,382Q, 382R, 382S, 382T, 382V, 382W, 382Y, 385E, 385P, 386K, 423N, 424H,424M, 424V, 426D, 426L, 427N, 429A, 429F, 429M, 430A, 430D, 430F, 430G,430H, 430I, 430K, 430L, 430M, 430N, 430P, 430Q, 430R, 430S, 430T, 430V,430W, 430Y, 431H, 431K, 431P, 432R, 432S, 438G, 438K, 438L, 438T, 438W,439E, 439H, 439Q, 440D, 440E, 440F, 440G, 440H, 440I, 440K, 440L, 440M,440Q, 440T, 440V or 442K. As above, the numbering of the residues in theheavy chain is that of the EU index (see Kabat et al., supra). Suchvariant Fc regions typically confer an altered effector function oraltered serum half-life upon the antibody to which the variant Fc regionis operably attached. Preferably the altered effector function is anincrease in ADCC, a decrease in ADCC, an increase in CDC, a decrease inCDC, an increase in Clq binding affinity, a decrease in Clq bindingaffinity, an increase in FcR (preferably FcRn) binding affinity or adecrease in FcR (preferably FcRn) binding affinity as compared to acorresponding Fc region that lacks such amino acid substitution(s).

Additional examples include variant Fc regions that comprise an aminoacid substitution at one or more of position(s) 221, 222, 224, 227, 228,230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244,245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 283, 285, 286, 288,290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 302, 313, 317, 318,320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335 336 and/or 428 (see, e.g., U.S. Pat. No. 7,662,925). In specificembodiments, the variant Fc region comprises at least one amino acidsubstitution selected from the group consisting of: P230A, E233D, L234E,L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q,S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T,K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D,S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V,L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R,and T335Y. In other specific embodiments, the variant Fc regioncomprises at least one amino acid substitution selected from the groupconsisting of: V264I, F243L/V264I, L328M, I332E, L328M/I332E,V264I/I332E, S298A/I332E, S239E/I332E, S239Q/I332E, S239E, A330Y, I332D,L328I/I332E, L328Q/I332E, V264T, V240I, V266I, S239D, S239D/I332D,S239D/I332E, S239D/I332N, S239D/I332Q, S239E/I332D, S239E/I332N,S239E/I332Q, S239N/I332D, S239N/I332E, S239Q/I332D, A330Y/I332E,V264I/A330Y/I332E, A330L/I332E, V264/A330L/I332E, L234E, L234Y, L234I,L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T,L328D/I332E, L328V/I332E, L328T/I332E, L328I/I332E, S239E/V264I/I332E,S239Q/V264I/I332E, S239E/V264/A330Y/I332E, S239D/A330Y/I332E,S239N/A330Y/I332E, S239D/A330L/I332E, S239N/A330L/I332E,V264/S298A/I332E, S239D/S298A/I332E, S239N/S298A/I332E,S239D/V264I/I332E, S239D/V264/S298A/I332E, S239D/V264I/A330L/I332E,S239D/I332E/A330I, P230A, P230A/E233D/I332E, E272Y, K274T, K274E, K274R,K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V,K326I, K326T, T335D, T335R, T335Y, V240/V266I, S239D/A330Y/I332E/L234I,S239D/A330Y/I332E/L235D, S239D/A330Y/I332E/V240I,S239D/A330Y/I332E/V264T, S239D/A330Y/I332E/K326E, andS239D/A330Y/I332E/K326T, In more specific embodiments, the variant Fcregion comprises a series of substitutions selected from the groupconsisting of: N297D/I332E, F241Y/F243Y/V262T/V264T/N297D/I332E,S239D/N297D/I332E, S239E/N297D/I332E, S239D/D265Y/N297D/I332E,S239D/D265H/N297D/I332E, V264E/N297D/I332E, Y296N/N297D/I332E,N297D/A330Y/I332E, S239D/D265V/N297D/I332E, S239D/D2651/N297D/I332E, andN297D/S298A/A330Y/I332E. In specific embodiments, the variant Fc regioncomprises an amino acid substitution at position 332 (using thenumbering of the EU index, Kabat et al., supra). Examples ofsubstitutions include 332A, 332D, 332E, 332F, 332G, 332H, 332K, 332L,332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W and 332Y. Thenumbering of the residues in the Fc region is that of the EU index ofKabat et al. Among other properties described herein, such variant Fcregions may have increased affinity for an FcγR, increased stability,and/or increased solubility, relative to a corresponding, wild-type Fcregion.

Further examples include variant Fc regions that comprise one or more ofthe following amino acid substitutions: 224N/Y, 225A, 228L, 230S, 239P,240A, 241L, 243S/L/G/H/I, 244L, 246E, 247L/A, 252T, 254T/P, 258K, 261Y,265V, 266A, 267G/N, 268N, 269K/G, 273A, 276D, 278H, 279M, 280N, 283G,285R, 288R, 289A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 305A,306I/F, 311R, 312N, 315D/K/S, 320R, 322E, 323A, 324T, 325S, 326E/R,332T, 333D/G, 335I, 338R, 339T, 340Q, 341E, 342R, 344Q, 347R, 351S,352A, 354A, 355W, 356G, 358T, 361D/Y, 362L, 364C, 365Q/P, 370R, 372L,377V, 378T, 383N, 389S, 390D, 391C, 393A, 394A, 399G, 404S, 408G, 409R,411I, 412A, 414M, 421S, 422I, 426F/P, 428T, 430K, 431S, 432P, 433P,438L, 439E/R, 440G, 441F, 442T, 445R, 446A, 447E, optionally where thevariant has altered recognition of an Fc ligand and/or altered effectorfunction compared with a parent Fc polypeptide, and wherein thenumbering of the residues is that of the EU index as in Kabat et al.Specific examples of these and related embodiments include variant Fcregions that comprise or consist of the following sets of substitutions:(1) N276D, R292Q, V305A, I377V, T394A, V412A and K439E; (2) P244L,K246E, D399G and K409R; (3) S304G, K320R, S324T, K326E and M358T; (4)F243S, P247L, D265V, V266A, S383N and T411I; (5) H224N, F243L, T393A andH433P; (6) V240A, S267G, G341E and E356G; (7) M252T, P291L, P352A,R355W, N390D, S408G, S426F and A431S; (8) P228L, T289A, L365Q, N389S andS440G; (9) F241L, V273A, K340Q and L441F; (10) F241L, T299A, I332T andM428T; (11) E269K, Y300H, Q342R, V422I and G446A; (12) T225A, R301c,S304G, D312N, N315D, L351S and N421S; (13) S254T, L306I, K326R andQ362L; (14) H224Y, P230S, V323A, E333D, K338R and S364C; (15) T335I,K414M and P445R; (16) T335I and K414M; (17) P247A, E258K, D280N, K288R,N297D, T299A, K322E, Q342R, S354A and L365P; (18) H268N, V279M, A339T,N361D and S426P; (19) C261Y, K290E, L306F, Q311R, E333G and Q438L; (20)E283G, N315K, E333G, R344Q, L365P and S442T; (21) Q347R, N361Y andK439R; (22) S239P, S254P, S267N, H285R, N315S, F372L, A378T, N390D,Y391C, F404S, E430K, L432P and K447E; and (23) E269G, Y278H, N325S andK370R, wherein the numbering of the residues is that of the EU index asin Kabat et al. (see, e.g., U.S. Application No. 2010/0184959).

Variant Fc regions can also have one or more mutated hinge regions, asdescribed, for example, in U.S. Application No. 2003/0118592. Forinstance, one or more cysteines in a hinge region can be deleted orsubstituted with a different amino acid. The mutated hinge region cancomprise no cysteine residues, or it can comprise 1, 2, or 3 fewercysteine residues than a corresponding, wild-type hinge region. In someembodiments, an Fc region having a mutated hinge region of this typeexhibits a reduced ability to dimerize, relative to a wild-type Ig hingeregion.

In particular embodiments, the Fc region comprises, consists, orconsists essentially of the Fc from human IgG1 or IgG4 (see, e.g.,Allberse and Schuurman, Immunology. 105:9-19, 2002), or a fragment orvariant thereof. Table F below provides exemplary sequences (CH1, hinge(underlined), CH2, and CH3 regions) from human IgG1 and IgG4. Examplesof variant IgG4 sequences that can be employed are described, forexample, in Peters et al., JBC. 287:24525-24533, 2012, and includesubstitutions at C227, C230, C127 (e.g., C127S), and C131 (e.g., C131S).Other variants that can be used include a L445P substitution in IgG4(denoted as IgG4-2) or a D356E and L358M substitution in IgG1, (denotedas IgGm(zf)).

TABLE F1 Exemplary IgG4 Fc Sequences SEQ ID Name Sequence NO: Wild-typeASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV 144 IgG4HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG4-ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV 145 M265Y,HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES S267T,T269EKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQED (YTE,PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK KabatCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK numbering)GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG4-S241PASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV 146 (KabatHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES numbering)KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK IgG1m(za)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV 147 GenBank:HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP AH007035.2KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK KappaRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG 148 Km3NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

As noted above, antibodies having altered Fc regions typically havealtered (e.g., improved, increased, decreased) pharmacokineticproperties relative to corresponding wild-type Fc region. Examples ofpharmacokinetic properties include stability or half-life,bioavailability (the fraction of a drug that is absorbed), tissuedistribution, volume of distribution (apparent volume in which a drug isdistributed immediately after it has been injected intravenously andequilibrated between plasma and the surrounding tissues), concentration(initial or steady-state concentration of drug in plasma), eliminationrate constant (rate at which drugs are removed from the body),elimination rate (rate of infusion required to balance elimination),area under the curve (AUC or exposure; integral of theconcentration-time curve, after a single dose or in steady state),clearance (volume of plasma cleared of the drug per unit time), C_(max)(peak plasma concentration of a drug after oral administration), t_(max)(time to reach C_(max)), C_(min) (lowest concentration that a drugreaches before the next dose is administered), and fluctuation (peaktrough fluctuation within one dosing interval at steady state).

In particular embodiments, an antibody or antigen-binding fragmentthereof has a biological half life at about pH 7.4, at about aphysiological pH, at about 25° C. or room temperature, and/or at about37° C. or human body temperature (e.g., in vivo, in serum, in a giventissue, in a given species such as rat, mouse, monkey, or human), ofabout or at least about 30 minutes, about 1 hour, about 2 hour, about 3hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours,about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours,about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90hours, about 96 hours, about 120 hours, or about 144 hours or more, orabout 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, orabout 5 weeks, or about 6 weeks or more, or any intervening half-life,including all ranges in between.

In some embodiments, an antibody or antigen-binding fragment thereof hasa T_(m) of about or at least about 60, 62, 64, 66, 68, 70, 72, 74, or75° C. In some embodiments, an antibody or antigen-binding fragmentthereof has a T_(m) of about 60° C. or greater.

In some embodiments, an antibody or antigen-binding fragment thereofconjugated to one or more cytotoxic or chemotherapeutic agents. Generalexamples of cytotoxic or chemotherapeutic agents include, withoutlimitation, alkylating agents, anti-metabolites, anthracyclines,anti-tumor antibiotics, platinums, type I topoisomerase inhibitors, typeII topoisomerase inhibitors, vinca alkaloids, and taxanes. Specificexamples of cytotoxic or chemotherapeutic agents include, withoutlimitation, cyclophosphamide, cilengitide, lomustine (CCNU), melphalan,procarbazine, carmustine (BCNU), enzastaurin, busulfan, daunorubicin,doxorubicin, gefitinib, erlotinib idarubicin, temozolomide, epirubicin,mitoxantrone, bleomycin, cisplatin, carboplatin, oxaliplatin,camptothecins, irinotecan, topotecan, amsacrine, etoposide, etoposidephosphate, teniposide, temsirolimus, everolimus, vincristine,vinblastine, vinorelbine, vindesine, CT52923, paclitaxel, imatinib,dasatinib, sorafenib, pazopanib, sunitnib, vatalanib, geftinib,erlotinib, AEE-788, dichoroacetate, tamoxifen, fasudil, SB-681323,semaxanib, donepizil, galantamine, memantine, rivastigmine, tacrine,rasigiline, naltrexone, lubiprostone, safinamide, istradefylline,pimavanserin, pitolisant, isradipine, pridopidine (ACR16),tetrabenazine, bexarotene, glatirimer acetate, fingolimod, andmitoxantrone, including pharmaceutically acceptable salts and acidsthereof. Further examples of cytotoxic or chemotherapeutic agentsinclude alkylating agents such as thiotepa, cyclophosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.,paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®., Rhne-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid derivatives such asTargretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukindiftitox); esperamicins; capecitabine; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

The antibodies or antigen-binding fragments thereof can be used in anyof the compositions, methods, and/or kits described herein, and combinedwith one or more of the immunotherapy agents described herein.

Additional Therapeutic Agents and Compositions

Immunotherapy Agents. Certain embodiments employ one or more cancerimmunotherapy agents. In certain instances, an immunotherapy agentmodulates the immune response of a subject, for example, to increase ormaintain a cancer-related or cancer-specific immune response, andthereby results in increased immune cell inhibition or reduction ofcancer cells. Exemplary immunotherapy agents include polypeptides, forexample, antibodies and antigen-binding fragments thereof, ligands, andsmall peptides, and mixtures thereof. Also include as immunotherapyagents are small molecules, cells (e.g., immune cells such as T-cells),various cancer vaccines, gene therapy or other polynucleotide-basedagents, including viral agents such as oncolytic viruses, and othersknown in the art. Thus, in certain embodiments, the cancer immunotherapyagent is selected from one or more of immune checkpoint modulatoryagents, cancer vaccines, oncolytic viruses, cytokines, and a cell-basedimmunotherapies.

In certain embodiments, the cancer immunotherapy agent is an immunecheckpoint modulatory agent. Particular examples include “antagonists”of one or more inhibitory immune checkpoint molecules, and “agonists” ofone or more stimulatory immune checkpoint molecules. Generally, immunecheckpoint molecules are components of the immune system that eitherturn up a signal (co-stimulatory molecules) or turn down a signal, thetargeting of which has therapeutic potential in cancer because cancercells can perturb the natural function of immune checkpoint molecules(see, e.g., Sharma and Allison, Science. 348:56-61, 2015; Topalian etal., Cancer Cell. 27:450-461, 2015; Pardoll, Nature Reviews Cancer.12:252-264, 2012). In some embodiments, the immune checkpoint modulatoryagent (e.g., antagonist, agonist) “binds” or “specifically binds” to theone or more immune checkpoint molecules, as described herein.

In particular embodiments, the immune checkpoint modulatory agent is apolypeptide or peptide. The terms “peptide” and “polypeptide” are usedinterchangeably herein, however, in certain instances, the term“peptide” can refer to shorter polypeptides, for example, polypeptidesthat consist of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids, including allintegers and ranges (e.g., 5-10, 8-12, 10-15) in between. Polypeptidesand peptides can be composed of naturally-occurring amino acids and/ornon-naturally occurring amino acids, as described herein

Antibodies are also included as polypeptides. Thus, in some embodiments,the immune checkpoint modulatory polypeptide agent is an antibody or“antigen-binding fragment thereof”, as described elsewhere herein.

In some embodiments, the agent is or comprises a “ligand,” for example,a natural ligand, of the immune checkpoint molecule. A “ligand” refersgenerally to a substance or molecule that forms a complex with a targetmolecule (e.g., biomolecule) to serve a biological purpose, and includesa “protein ligand,” which generally produces a signal by binding to asite on a target molecule or target protein. Thus, certain agents areprotein ligands that, in nature, bind to an immune checkpoint moleculeand produce a signal. Also included are “modified ligands,” for example,protein ligands that are fused to a pharmacokinetic modifier, forexample, an Fc region derived from an immunoglobulin.

The binding properties of polypeptides can be quantified using methodswell known in the art (see Davies et al., Annual Rev. Biochem.59:439-473, 1990). In some embodiments, a polypeptide specifically bindsto a target molecule, for example, an immune checkpoint molecule or anepitope thereof, with an equilibrium dissociation constant that is aboutor ranges from about ≤10-7 to about 10-8 M. In some embodiments, theequilibrium dissociation constant is about or ranges from about ≤10-9 Mto about ≤10-10 M. In certain illustrative embodiments, the polypeptidehas an affinity (Kd or EC₅₀) for a target described herein (to which itspecifically binds) of about, at least about, or less than about, 0.01,0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 40, or 50 nM.

In some embodiments, the agent is a “small molecule,” which refers to anorganic compound that is of synthetic or biological origin(biomolecule), but is typically not a polymer. Organic compounds referto a large class of chemical compounds whose molecules contain carbon,typically excluding those that contain only carbonates, simple oxides ofcarbon, or cyanides. A “biomolecule” refers generally to an organicmolecule that is produced by a living organism, including largepolymeric molecules (biopolymers) such as peptides, polysaccharides, andnucleic acids as well, and small molecules such as primary secondarymetabolites, lipids, phospholipids, glycolipids, sterols, glycerolipids,vitamins, and hormones. A “polymer” refers generally to a large moleculeor macromolecule composed of repeating structural units, which aretypically connected by covalent chemical bond.

In certain embodiments, a small molecule has a molecular weight of aboutor less than about 1000-2000 Daltons, typically between about 300 and700 Daltons, and including about or less than about 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 500, 650, 600, 750, 700, 850, 800,950, 1000 or 2000 Daltons.

Certain small molecules can have the “specific binding” characteristicsdescribed for herein polypeptides such as antibodies. For instance, insome embodiments a small molecule specifically binds to a target, forexample, an immune checkpoint molecule, with a binding affinity (Kd orEC₅₀) of about, at least about, or less than about, 0.01, 0.05, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 40, or 50 nM.

In some embodiments, the immune checkpoint modulatory agent is anantagonist or inhibitor of one or more inhibitory immune checkpointmolecules. Exemplary inhibitory immune checkpoint molecules includeProgrammed Death-Ligand 1 (PD-L1), Programmed Death-Ligand 2 (PD-L2),Programmed Death 1 (PD-1), Cytotoxic T-Lymphocyte-Associated protein 4(CTLA-4), Indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase(TDO), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3),Lymphocyte Activation Gene-3 (LAG-3), V-domain Ig suppressor of T cellactivation (VISTA), B and T Lymphocyte Attenuator (BTLA), CD160, andT-cell immunoreceptor with Ig and ITIM domains (TIGIT).

In certain embodiments, the agent is a PD-1 (receptor) antagonist orinhibitor, the targeting of which has been shown to restore immunefunction in the tumor environment (see, e.g., Phillips et al., IntImmunol. 27:39-46, 2015). PD-1 is a cell surface receptor that belongsto the immunoglobulin superfamily and is expressed on T cells and pro-Bcells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 functionsas an inhibitory immune checkpoint molecule, for example, by reducing orpreventing the activation of T-cells, which in turn reduces autoimmunityand promotes self-tolerance. The inhibitory effect of PD-1 isaccomplished at least in part through a dual mechanism of promotingapoptosis in antigen specific T-cells in lymph nodes while also reducingapoptosis in regulatory T cells (suppressor T cells). Some examples ofPD-1 antagonists or inhibitors include an antibody or antigen-bindingfragment or small molecule that specifically binds to PD-1 and reducesone or more of its immune-suppressive activities, for example, itsdownstream signaling or its interaction with PD-L1. Specific examples ofPD-1 antagonists or inhibitors include the antibodies nivolumab,pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and pidilizumab, andantigen-binding fragments thereof (see, e.g., U.S. Pat. Nos. 8,008,449;8,993,731; 9,073,994; 9,084,776; 9,102,727; 9,102,728; 9,181,342;9,217,034; 9,387,247; 9,492,539; 9,492,540; and U.S. Application Nos.2012/0039906; 2015/0203579).

In some embodiments, the agent is a PD-L1 antagonist or inhibitor. Asnoted above, PD-L1 is one of the natural ligands for the PD-1 receptor.General examples of PD-L1 antagonists or inhibitors include an antibodyor antigen-binding fragment or small molecule that specifically binds toPD-L1 and reduces one or more of its immune-suppressive activities, forexample, its binding to the PD-1 receptor. Specific examples of PD-L1antagonists include the antibodies atezolizumab (MPDL3280A), avelumab(MSB0010718C), and durvalumab (MEDI4736), and antigen-binding fragmentsthereof (see, e.g., U.S. Pat. Nos. 9,102,725; 9,393,301; 9,402,899;9,439,962).

In some embodiments, the agent is a PD-L2 antagonist or inhibitor. Asnoted above, PD-L2 is one of the natural ligands for the PD-1 receptor.General examples of PD-L2 antagonists or inhibitors include an antibodyor antigen-binding fragment or small molecule that specifically binds toPD-L2 and reduces one or more of its immune-suppressive activities, forexample, its binding to the PD-1 receptor.

In some embodiments, the agent is a CTLA-4 antagonist or inhibitor.CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), alsoknown as CD152 (cluster of differentiation 152), is a protein receptorthat functions as an inhibitory immune checkpoint molecule, for example,by transmitting inhibitory signals to T-cells when it is bound to CD80or CD86 on the surface of antigen-presenting cells. General examplesCTLA-4 antagonists or inhibitors include an antibody or antigen-bindingfragment or small molecule that specifically binds to CTLA-4. Particularexamples include the antibodies ipilimumab and tremelimumab, andantigen-binding fragments thereof. At least some of the activity ofipilimumab is believed to be mediated by antibody-dependentcell-mediated cytotoxicity (ADCC) killing of suppressor Tregs thatexpress CTLA-4.

In some embodiments, the agent is an IDO antagonist or inhibitor, or aTDO antagonist or inhibitor. IDO and TDO are tryptophan catabolicenzymes with immune-inhibitory properties. For example, IDO is known tosuppress T-cells and NK cells, generate and activate Tregs andmyeloid-derived suppressor cells, and promote tumor angiogenesis.General examples of IDO and TDO antagonists or inhibitors include anantibody or antigen-binding fragment or small molecule that specificallybinds to IDO or TDO (see, e.g., Platten et al., Front Immunol. 5: 673,2014) and reduces or inhibits one or more immune-suppressive activities.Specific examples of IDO antagonists or inhibitors include indoximod(NLG-8189), 1-methyl-tryptophan (1MT), β-Carboline (norharmane;9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, e.g.,Sheridan, Nature Biotechnology. 33:321-322, 2015). Specific examples ofTDO antagonists or inhibitors include 680C91 and LM10 (see, e.g.,Pilotte et al., PNAS USA. 109:2497-2502, 2012).

In some embodiments, the agent is a TIM-3 antagonist or inhibitor.T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3) is expressed onactivated human CD4+ T-cells and regulates Th1 and Th17 cytokines. TIM-3also acts as a negative regulator of Th1/Tc1 function by triggering celldeath upon interaction with its ligand, galectin-9. TIM-3 contributes tothe suppressive tumor microenvironment and its overexpression isassociated with poor prognosis in a variety of cancers (see, e.g., Li etal., Acta Oncol. 54:1706-13, 2015). General examples of TIM-3antagonists or inhibitors include an antibody or antigen-bindingfragment or small molecule that specifically binds to TIM-3 and reducesor inhibits one or more of its immune-suppressive activities.

In some embodiments, the agent is a LAG-3 antagonist or inhibitor.Lymphocyte Activation Gene-3 (LAG-3) is expressed on activated T-cells,natural killer cells, B-cells and plasmacytoid dendritic cells. Itnegatively regulates cellular proliferation, activation, and homeostasisof T-cells, in a similar fashion to CTLA-4 and PD-1 (see, e.g., Workmanand Vignali. European Journal of Immun. 33: 970-9, 2003; and Workman etal., Journal of Immun. 172: 5450-5, 2004), and has been reported to playa role in Treg suppressive function (see, e.g., Huang et al., Immunity.21: 503-13, 2004). LAG3 also maintains CD8+ T-cells in a tolerogenicstate and combines with PD-1 to maintain CD8 T-cell exhaustion. Generalexamples of LAG-3 antagonists or inhibitors include an antibody orantigen-binding fragment or small molecule that specifically binds toLAG-3 and inhibits one or more of its immune-suppressive activities.Specific examples include the antibody BMS-986016, and antigen-bindingfragments thereof.

In some embodiments, the agent is a VISTA antagonist or inhibitor.V-domain Ig suppressor of T cell activation (VISTA) is primarilyexpressed on hematopoietic cells and is an inhibitory immune checkpointregulator that suppresses T-cell activation, induces Foxp3 expression,and is highly expressed within the tumor microenvironment where itsuppresses anti-tumor T cell responses (see, e.g., Lines et al., CancerRes. 74:1924-32, 2014). General examples of VISTA antagonists orinhibitors include an antibody or antigen-binding fragment or smallmolecule that specifically binds to VISTA and reduces one or more of itsimmune-suppressive activities.

In some embodiments, the agent is a BTLA antagonist or inhibitor. B- andT-lymphocyte attenuator (BTLA; CD272) expression is induced duringactivation of T-cells, and it inhibits T-cells via interaction withtumor necrosis family receptors (TNF-R) and B7 family of cell surfacereceptors. BTLA is a ligand for tumor necrosis factor (receptor)superfamily, member 14 (TNFRSF14), also known as herpes virus entrymediator (HVEM). BTLA-HVEM complexes negatively regulate T-cell immuneresponses, for example, by inhibiting the function of human CD8+cancer-specific T-cells (see, e.g., Derre et al., J Clin Invest120:157-67, 2009). General examples of BTLA antagonists or inhibitorsinclude an antibody or antigen-binding fragment or small molecule thatspecifically binds to BTLA-4 and reduce one or more of itsimmune-suppressive activities.

In some embodiments, the agent is an HVEM antagonist or inhibitor, forexample, an antagonist or inhibitor that specifically binds to HVEM andinterferes with its interaction with BTLA or CD160. General examples ofHVEM antagonists or inhibitors include an antibody or antigen-bindingfragment or small molecule that specifically binds to HVEM, optionallyreduces the HVEM/BTLA and/or HVEM/CD160 interaction, and thereby reducesone or more of the immune-suppressive activities of HVEM.

In some embodiments, the agent is a CD160 antagonist or inhibitor, forexample, an antagonist or inhibitor that specifically binds to CD160 andinterferes with its interaction with HVEM. General examples of CD160antagonists or inhibitors include an antibody or antigen-bindingfragment or small molecule that specifically binds to CD160, optionallyreduces the CD160/HVEM interaction, and thereby reduces or inhibits oneor more of its immune-suppressive activities.

In some embodiments, the agent is a TIGIT antagonist or inhibitor. Tcell Ig and ITIM domain (TIGIT) is a co-inhibitory receptor that isfound on the surface of a variety of lymphoid cells, and suppressesantitumor immunity, for example, via Tregs (Kurtulus et al., J ClinInvest. 125:4053-4062, 2015). General examples of TIGIT antagonists orinhibitors include an antibody or antigen-binding fragment or smallmolecule that specifically binds to TIGIT and reduce one or more of itsimmune-suppressive activities (see, e.g., Johnston et al., Cancer Cell.26:923-37, 2014).

In certain embodiments, the immune checkpoint modulatory agent is anagonist of one or more stimulatory immune checkpoint molecules.Exemplary stimulatory immune checkpoint molecules include OX40, CD40,Glucocorticoid-Induced TNFR Family Related Gene (GITR), CD137 (4-1BB),CD27, CD28, CD226, and Herpes Virus Entry Mediator (HVEM).

In some embodiments, the agent is an OX40 agonist. OX40 (CD134) promotesthe expansion of effector and memory T cells, and suppresses thedifferentiation and activity of T-regulatory cells (see, e.g., Croft etal., Immunol Rev. 229:173-91, 2009). Its ligand is OX40L (CD252). SinceOX40 signaling influences both T-cell activation and survival, it playsa key role in the initiation of an anti-tumor immune response in thelymph node and in the maintenance of the anti-tumor immune response inthe tumor microenvironment. General examples of OX40 agonists include anantibody or antigen-binding fragment or small molecule or ligand thatspecifically binds to OX40 and increases one or more of itsimmunostimulatory activities. Specific examples include OX86, OX-40L,Fc-OX40L, GSK3174998, MEDI0562 (a humanized OX40 agonist), MEDI6469(murine OX4 agonist), and MEDI6383 (an OX40 agonist), andantigen-binding fragments thereof.

In some embodiments, the agent is a CD40 agonist. CD40 is expressed onantigen-presenting cells (APC) and some malignancies. Its ligand isCD40L (CD154). On APC, ligation results in upregulation of costimulatorymolecules, potentially bypassing the need for T-cell assistance in anantitumor immune response. CD40 agonist therapy plays an important rolein APC maturation and their migration from the tumor to the lymph nodes,resulting in elevated antigen presentation and T cell activation.Anti-CD40 agonist antibodies produce substantial responses and durableanticancer immunity in animal models, an effect mediated at least inpart by cytotoxic T-cells (see, e.g., Johnson et al. Clin Cancer Res.21: 1321-1328, 2015; and Vonderheide and Glennie, Clin Cancer Res.19:1035-43, 2013). General examples of CD40 agonists include an antibodyor antigen-binding fragment or small molecule or ligand thatspecifically binds to CD40 and increases one or more of itsimmunostimulatory activities. Specific examples include CP-870,893,dacetuzumab, Chi Lob 7/4, ADC-1013, CD40L, rhCD40L, and antigen-bindingfragments thereof.

In some embodiments, the agent is a GITR agonist. Glucocorticoid-InducedTNFR family Related gene (GITR) increases T cell expansion, inhibits thesuppressive activity of Tregs, and extends the survival of T-effectorcells. GITR agonists have been shown to promote an anti-tumor responsethrough loss of Treg lineage stability (see, e.g., Schaer et al., CancerImmunol Res. 1:320-31, 2013). These diverse mechanisms show that GITRplays an important role in initiating the immune response in the lymphnodes and in maintaining the immune response in the tumor tissue. Itsligand is GITRL. General examples of GITR agonists include an antibodyor antigen-binding fragment or small molecule or ligand thatspecifically binds to GITR and increases one or more of itsimmunostimulatory activities. Specific examples include GITRL,INCAGN01876, DTA-1, MEDI1873, and antigen-binding fragments thereof.

In some embodiments, the agent is a CD137 agonist. CD137 (4-1BB) is amember of the tumor necrosis factor (TNF) receptor family, andcrosslinking of CD137 enhances T-cell proliferation, IL-2 secretion,survival, and cytolytic activity. CD137-mediated signaling also protectsT-cells such as CD8+ T-cells from activation-induced cell death. Generalexamples of CD137 agonists include an antibody or antigen-bindingfragment or small molecule or ligand that specifically binds to CD137and increases one or more of its immunostimulatory activities. Specificexamples include the CD137 (or 4-1BB) ligand (see, e.g., Shao andSchwarz, J Leukoc Biol. 89:21-9, 2011) and the antibody utomilumab,including antigen-binding fragments thereof.

In some embodiments, the agent is a CD27 agonist. Stimulation of CD27increases antigen-specific expansion of naïve T cells and contributes toT-cell memory and long-term maintenance of T-cell immunity. Its ligandis CD70. The targeting of human CD27 with an agonist antibody stimulatesT-cell activation and antitumor immunity (see, e.g., Thomas et al.,Oncoimmunology. 2014; 3:e27255. doi:10.4161/onci.27255; and He et al., JImmunol. 191:4174-83, 2013). General examples of CD27 agonists includean antibody or antigen-binding fragment or small molecule or ligand thatspecifically binds to CD27 and increases one or more of itsimmunostimulatory activities. Specific examples include CD70 and theantibodies varlilumab and CDX-1127 (1F5), including antigen-bindingfragments thereof.

In some embodiments, the agent is a CD28 agonist. CD28 is constitutivelyexpressed CD4+ T cells some CD8+ T cells. Its ligands include CD80 andCD86, and its stimulation increases T-cell expansion. General examplesof CD28 agonists include an antibody or antigen-binding fragment orsmall molecule or ligand that specifically binds to CD28 and increasesone or more of its immunostimulatory activities. Specific examplesinclude CD80, CD86, the antibody TAB08, and antigen-binding fragmentsthereof.

In some embodiments, the agent is CD226 agonist. CD226 is a stimulatingreceptor that shares ligands with TIGIT, and opposite to TIGIT,engagement of CD226 enhances T-cell activation (see, e.g., Kurtulus etal., J Clin Invest. 125:4053-4062, 2015; Bottino et al., J Exp Med.1984:557-567, 2003; and Tahara-Hanaoka et al., Int Immunol. 16:533-538,2004). General examples of CD226 agonists include an antibody orantigen-binding fragment or small molecule or ligand (e.g., CD112,CD155) that specifically binds to CD226 and increases one or more of itsimmunostimulatory activities.

In some embodiments, the agent is an HVEM agonist. Herpesvirus entrymediator (HVEM), also known as tumor necrosis factor receptorsuperfamily member 14 (TNFRSF14), is a human cell surface receptor ofthe TNF-receptor superfamily. HVEM is found on a variety of cellsincluding T-cells, APCs, and other immune cells. Unlike other receptors,HVEM is expressed at high levels on resting T-cells and down-regulatedupon activation. It has been shown that HVEM signaling plays a crucialrole in the early phases of T-cell activation and during the expansionof tumor-specific lymphocyte populations in the lymph nodes. Generalexamples of HVEM agonists include an antibody or antigen-bindingfragment or small molecule or ligand that specifically binds to HVEM andincreases one or more of its immunostimulatory activities.

In certain embodiments, the cancer immunotherapy agent is a cancervaccine. Exemplary cancer vaccines include Oncophage, humanpapillomavirus HPV vaccines such Gardasil or Cervarix, hepatitis Bvaccines such as Engerix-B, Recombivax HB, or Twinrix, and sipuleucel-T(Provenge). In some embodiments, the cancer vaccine comprises orutilizes one or more cancer antigens, or cancer-associate d antigens.Exemplary cancer antigens include, without limitation, human Her2/neu,Her1/EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20,CD22, CD23 (IgE Receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13,vascular endothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2,VEGFR-3, NRP2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74,CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin,vimentin, insulin-like growth factor 1 receptor (IGF-1R),alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonicanhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C,NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor1, transmembrane glycoprotein NMB, fibroblast activation protein alpha(FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125),phosphatidylserine, prostate-specific membrane antigen (PSMA), NR-LU-13antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b(TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), and mesothelin.

In certain embodiments, the cancer immunotherapy agent is an oncolyticvirus. An oncolytic virus is a virus that preferentially infects andkills cancer cells. Included are naturally-occurring and man-made orengineered oncolytic viruses. Most oncolytic viruses are engineered fortumor selectivity, although there are naturally-occurring examples suchas Reovirus and the SVV-001 Seneca Valley virus. General examples ofoncolytic viruses include VSV, Poliovirus, Reovirus, Senecavirus, andRIGVIR, and engineered versions thereof. Non-limiting examples ofoncolytic viruses include herpes simplex virus (HSV) and engineeredversion thereof, talimogene laherparepvec (T-VEC), coxsackievirus A21(CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus(NTX-010), Senecavirus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102(Ad5/3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401, among others.

In certain embodiments, the cancer immunotherapy agent is a cytokine.Exemplary cytokines include interferon (IFN)-α, IL-2, IL-12, IL-7,IL-21, and Granulocyte-macrophage colony-stimulating factor (GM-CSF).

In certain embodiments, the cancer immunotherapy agent is cell-basedimmunotherapy, for example, a T-cell based adoptive immunotherapy. Insome embodiments, the cell-based immunotherapy comprises cancerantigen-specific T-cells, optionally ex vivo-derived T-cells. In someembodiments, the cancer antigen-specific T-cells are selected from oneor more of chimeric antigen receptor (CAR)-modified T-cells, and T-cellReceptor (TCR)-modified T-cells, tumor infiltrating lymphocytes (TILs),and peptide-induced T-cells. In specific embodiments, the CAR-modifiedT-cell is targeted against CD-19 (see, e.g., Maude et al., Blood.125:4017-4023, 2015).

In certain instances, the cancer to be treated associates with thecancer antigen, that is, the cancer antigen-specific T-cells aretargeted against or enriched for at least one antigen that is known toassociate with the cancer to be treated. In some embodiments, the cancerantigen is selected from one or more of CD19, human Her2/neu, Her1/EGFreceptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD20, CD22, CD23 (IgEReceptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelialgrowth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30, CD33, CD37,CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4,HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1(IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA),guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrinα5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblastactivation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (orCA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA),NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamilymember 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derivedgrowth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1,protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3(PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (adisialoganglioside expressed on tumors of neuroectodermal origin),glypican-3 (GPC3), and mesothelin.

Additional exemplary cancer antigens include 5T4, 707-AP, 9D7, AFP,AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin,alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m,B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m,CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m,cathepsin B, cathepsin L, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28,CML66, COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6,Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN,EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1,EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5,GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2,hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R171, HLA-A11/m, HLA-A2/m, HNE,homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M,HST-2, hTERT, iCE, IGF-1 R, IL-13Ra2, IL-2R, IL-5, immature lamininreceptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m,KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3,MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B1 6, MAGE-B1 7, MAGE-C1,MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1,MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrixprotein 22, MCI R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class1/m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m,NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-B, NY-ESO-1, OA1,OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, pi 5, p190minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE,PDEF, Pim-1 Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein,proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,RHAMM/CD1 68, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC,SIRT2/m, Sp1 7, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1,survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP,TEL-AML1, TGF-beta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1,TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGFR, VEGFR-2/FLK-1, andWT1. Certain preferred antigens include p53, CA125, EGFR, Her2/neu,hTERT, PAP, MAGE-A1, MAGE-A3, Mesothelin, MUC-1, GP100, MART-1,Tyrosinase, PSA, PSCA, PSMA, STEAP-1, Ras, CEA and WT1, and morepreferably PAP, MAGE-A3, WT1, and MUC-1.

In some embodiments the antigen is selected from MAGE-A1 (e.g., MAGE-A1according to accession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g.,MAGE-A6 according to accession number NM_005363), MAGE-C1, MAGE-C2,melan-A (e.g., melan-A according to accession number NM_00551 1), GP100(e.g., GP100 according to accession number M77348), tyrosinase (e.g.,tyrosinase according to accession number NM_000372), survivin (e.g.,survivin according to accession number AF077350), CEA (e.g., CEAaccording to accession number NM_004363), Her-2/neu (e.g., Her-2/neuaccording to accession number M1 1730), WT1 (e.g., WT1 according toaccession number NM_000378), PRAME (e.g., PRAME according to accessionnumber NM_006115), EGFRI (epidermal growth factor receptor 1) (e.g.,EGFRI (epidermal growth factor receptor 1) according to accession numberAF288738), MUC1, mucin-1 (e.g., mucin-1 according to accession numberNM_002456), SEC61 G (e.g., SEC61 G according to accession numberNM_014302), hTERT (e.g., hTERT accession number NM_198253), 5T4 (e.g.,5T4 according to accession number NM_006670), TRP-2 (e.g., TRP-2according to accession number NM_001922), STEAP1 (Six-transmembraneepithelial antigen of prostate 1), PSCA, PSA, PSMA, etc.

In some embodiments, the cancer antigen is selected from PCA, PSA, PSMA,STEAP, and optionally MUC-1, including fragments, variants, andderivatives thereof. In some embodiments, the cancer antigen selectedfrom NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and optionally MUC-1,including fragments, variants, and derivatives thereof.

In some instances, cancer antigens encompass idiotypic antigensassociated with a cancer or tumor disease, particularly lymphoma or alymphoma associated disease, for example, wherein the idiotypic antigenis an immunoglobulin idiotype of a lymphoid blood cell or a T cellreceptor idiotype of a lymphoid blood cell.

In some instances, the cancer antigen-specific T-cells are selected fromone or more of chimeric antigen receptor (CAR)-modified T-cells (e.g.,targeted against a cancer antigen), and T-cell Receptor (TCR)-modifiedT-cells, tumor infiltrating lymphocytes (TILs), and peptide-inducedT-cells.

The skilled artisan will appreciate that the various cancerimmunotherapy agents described herein can be combined with any one ormore of the various anti-NRP2 antibodies (including antigen-bindingfragments thereof) described herein, and used according to any one ormore of the methods or compositions described herein.

Chemotherapeutic Agents. Certain embodiments employ one or morechemotherapeutic agents, for example, small molecule chemotherapeuticagents. Non-limiting examples of chemotherapeutic agents includealkylating agents, anti-metabolites, cytotoxic antibiotics,topoisomerase inhibitors (type 1 or type II), an anti-microtubuleagents, among others.

Examples of alkylating agents include nitrogen mustards (e.g.,mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil,ifosfamide, and busulfan), nitrosoureas (e.g., N-Nitroso-N-methylurea(MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU),fotemustine, and streptozotocin), tetrazines (e.g., dacarbazine,mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mytomycin,and diaziquone (AZQ)), cisplatins and derivatives thereof (e.g.,carboplatin and oxaliplatin), and non-classical alkylating agents(optionally procarbazine and hexamethylmelamine).

Examples of anti-metabolites include anti-folates (e.g., methotrexateand pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil andcapecitabine), deoxynucleoside analogues (e.g., ancitabine, enocitabine,cytarabine, gemcitabine, decitabine, azacitidine, fludarabine,nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), andthiopurines (e.g., thioguanine and mercaptopurine);

Examples of cytotoxic antibiotics include anthracyclines (e.g.,doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin,aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone,and actinomycin. Examples of topoisomerase inhibitors includecamptothecin, irinotecan, topotecan, etoposide, doxorubicin,mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin.

Examples of anti-microtubule agents include taxanes (e.g., paclitaxeland docetaxel) and vinca alkaloids (e.g., vinblastine, vincristine,vindesine, vinorelbine).

The skilled artisan will appreciate that the various chemotherapeuticagents described herein can be combined with any one or more of thevarious anti-NRP2 antibodies (including antigen-binding fragmentsthereof) described herein, and used according to any one or more of themethods or compositions described herein.

Hormonal Therapeutic Agents. Certain embodiments employ at least onehormonal therapeutic agent. General examples of hormonal therapeuticagents include hormonal agonists and hormonal antagonists. Particularexamples of hormonal agonists include progestogen (progestin),corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone),insulin like growth factors, VEGF derived angiogenic and lymphangiogenicfactors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2),fibroblast growth factor (FGF), galectin, hepatocyte growth factor(HGF), platelet derived growth factor (PDGF), transforming growth factor(TGF)-beta, androgens, estrogens, and somatostatin analogs. Examples ofhormonal antagonists include hormone synthesis inhibitors such asaromatase inhibitors and gonadotropin-releasing hormone (GnRH)s agonists(e.g., leuprolide, goserelin, triptorelin, histrelin) including analogsthereof. Also included are hormone receptor antagonist such as selectiveestrogen receptor modulators (SERMs; e.g., tamoxifen, raloxifene,toremifene) and anti-androgens (e.g., flutamide, bicalutamide,nilutamide).

Also included are hormonal pathway inhibitors such as antibodiesdirected against hormonal receptors. Examples include inhibitors of theIGF receptor (e.g., IGF-IR1) such as cixutumumab, dalotuzumab,figitumumab, ganitumab, istiratumab, and robatumumab; inhibitors of thevascular endothelial growth factor receptors 1, 2 or 3 (VEGFR, VEGFR2 orVEGFR3) such as alacizumab pegol, bevacizumab, icrucumab, ramucirumab;inhibitors of the TGF-beta receptors R1, R2, and R3 such as fresolimumaband metelimumab; inhibitors of c-Met such as naxitamab; inhibitors ofthe EGF receptor such as cetuximab, depatuxizumab mafodotin, futuximab,imgatuzumab, laprituximab emtansine, matuzumab, modotuximab,necitumumab, nimotuzumab, panitumumab, tomuzotuximab, and zalutumumab;inhibitors of the FGF receptor such as aprutumab ixadotin andbemarituzumab; and inhibitors of the PDGF receptor such as olaratumaband tovetumab.

The skilled artisan will appreciate that the various hormonaltherapeutic agents described herein can be combined with any one or moreof the various anti-NRP2 antibodies (including antigen-binding fragmentsthereof) described herein, and used according to any one or more of themethods or compositions described herein.

Kinase Inhibitors. Certain embodiments employ at least one kinaseinhibitor, including tyrosine kinase inhibitors. Examples of kinaseinhibitors include, without limitation, adavosertib, afanitib,aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab,cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib,fostamitinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib,mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib,ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656,tofacitinib, trastuzumab, vandetanib, and vemuafenib. Exemplary PI3kinase inhibitors include alpelisib, buparlisib, copanlisib, CUDC-907,dactolisib, duvelisib, GNE-477, idelasib, IPI-549, LY294002, ME-401,perifosine, PI-103, pictilisib, PWT33597, RP6503, taselisib, umbralisib,voxtalisib, wortmannin, and XL147 The skilled artisan will appreciatethat the various kinase inhibitors described herein can be combined withany one or more of the various anti-NRP2 antibodies (includingantigen-binding fragments thereof) described herein, and used accordingto any one or more of the methods or compositions described herein.

Methods of Use and Therapeutic Compositions

Embodiments of the present disclosure relate in part to the discoverythat human histidyl-tRNA synthetase (HRS) polypeptides have unexpectedbiological properties which are relevant to treating a broad range ofdiseases and conditions, and that certain of these properties relate tothe interactions between HRS and human neuropilin 2 (NRP2). Accordingly,antibodies directed against human NRP2, which interfere with the bindingbetween NRP2 and NRP2 ligands and/or modulate NRP2 receptor signalingcan be used as standalone therapies in the treatment of diseases,including NRP2-associated diseases, or in combination with othertherapeutic agents as described herein.

Certain embodiments therefore include methods of treating, amelioratingthe symptoms of, and/or reducing the progression of, a disease orcondition in a subject in need thereof, comprising administering to thesubject at least one antibody or antigen-binding fragment thereof thatspecifically binds to a human neuropilin-2 (NRP2) polypeptide. In someinstances, the at least one antibody or antigen-binding fragment thereofantagonizes the binding/signaling activity between the NRP2 polypeptideand the at least one NRP2 ligand. In some instances, the at least oneantibody or antigen-binding fragment thereof mimics or otherwiseenhances one or more signaling activities of the NRP2/NRP2 ligandinteraction, for example, by acting as an agonist antibody.

In some embodiments the anti-NRP2 antibody binds selectively to theNRP2a isoform (e.g., variants 1, 2, and/or 3 of Table N1) of NRP2, anddoes not substantially bind to the NRP2b isoform (e.g., variants 4and/or 5 of Table N1). In some embodiments the anti-NRP2 antibody bindsselectively to the NRP2b isoform (e.g., variants 4 and/or 5 of TableN1), and does not substantially bind to the NRP2a isoform (e.g.,variants 1, 2, and/or 3 of Table N1). Such binding characteristics canapply to any one or more of the embodiments described herein.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof selectively modulates the binding and/or signaling ofVEGF-C or related NRP2 ligands to or via an NRP2 polypeptide. In someaspects, such antibodies or antigen-binding fragment thereof do notsubstantially block the interaction of semaphorins to an NRP2polypeptide. In some aspects, such antibodies or antigen-bindingfragment thereof are antagonistic antibodies with respect to VEGF-Csignaling via an NRP2 polypeptide. In some embodiments, such antibodiesare agonistic antibodies with respect to semaphorin 3F signaling via anNRP2 polypeptide. In some embodiments, the at least one antibody orantigen-binding fragment thereof antagonizes the binding/signalingactivity between an NRP2 polypeptide and VEGFR2 or VEGFR3 withoutsubstantially modulating the binding/signaling activity between an NRP2polypeptide and a plexin receptor and/or a semaphorin.

Some embodiments comprise administering at least one anti-NRP2 antibodyto a subject in an amount and at a frequency sufficient to achieve anaverage, sustained blood plasma concentration of soluble NRP2 of aboutor less than about 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pm, 40 pM,30 pM, 20 pM, or 10 pM. Certain embodiments comprise administering atleast one anti-NRP2 antibody in an amount and at a frequency sufficientto achieve a reduction in the circulating levels of HRS:NRP2 complexes,for example, a reduction of about or at least about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, 95, 99, or 100%. Some embodimentsinclude administering the at least one anti-NRP2 antibody in an amountand at a frequency sufficient to achieve a steady state concentration,or average circulating concentration, of the at least one anti-NRP2antibody of between about 1 nM and about 1 pM, between about 1 nM andabout 100 nM, between about 1 nM and about 10 nM, or between about 1 nMand about 3 pM.

In certain embodiments, the disease is or condition is anNRP2-associated disease or condition. In some embodiments, theNRP2-associated disease or condition is selected from one or more ofcancer and diseases and pathways associated with cancer, includingcancer cell growth, initiation, migration, adhesion, invasion,chemoresistance, and/or metastasis; diseases associated withinflammation, autoimmunity, and related inflammatory diseases, includingdiseases associated with inappropriate immune cell activation ormigration such as Graft versus host disease (GVHD); diseases associatedwith lymphatic development, lymphangiogenesis, and lymphatic damage,including, for example, edema, lymphedema, secondary lymphedema,inappropriate fat absorption and deposition, excess fat deposition, andvascular permeability; diseases associated with infections, includinglatent infections; diseases associated with allergic disorders/diseases,allergic responses, including, for example, chronic obstructivepulmonary disorder (COPD), neutrophilic asthma, antineutrophilcytoplasmic antibody (ANCA)-associated systemic vasculitis, systemiclupus erythematosus, rheumatoid arthritis, inflammasome-relateddiseases, and skin-related neutrophil-mediated diseases such as pyodermagangrenosum; diseases associated with granulomatous inflammatorydiseases, including sarcoidosis and granulomas; diseases associated withfibrosis including fibrotic diseases, fibrosis, endothelial tomesenchymal transition (EMT), and wound healing; diseases associatedwith inappropriate smooth muscle contractility, smooth musclecompensation and decompensation, and inappropriate vascular smoothmuscle cell migration and adhesion; diseases associated withinappropriate autophagy, phagocytosis, and efferocytosis; diseasesassociated with neuronal diseases, peripheral nervous system remodeling,and pain perception; and diseases associated with bone development andbone remodeling.

In some embodiments, the disease is a cancer. Here, upregulation of NRP2expression is associated with tumorigenesis, and in particular tumormetastasis and development of chemoresistance, and is correlated withmore aggressive disease in several tumor types. Moreover, thesemaphorin/plexin/neuropilin signaling axis influences many of thehallmarks of cancer (see, for example, Franzolin and Tamagnone Int. J.Mol. Sci. 20, 377; doi:10.3390/ijms20020377, 2019); Nasarre et al.,OncoTargets and Therapy 2014:7 1663-1687; Neufeld et al., Cold SpringHarb Perspect Med. 2:a006718, 2012). For example, Semaphorin 3F(SEMA3F), which was initially identified as tumor suppressor gene inlung cancer, and is consistently downregulated in highly metastatictumor cells, including prostate, bladder, and melanoma cells in vitroand in vivo. Consistent with these studies, increased expression of NRP2in prostate cancer cells is also induced by the loss of the tumorsuppressor gene phosphatase and tensin homolog (PTEN), and itsexpression correlates with Gleason grade (see, for example, Zhao et al.,Thoracic Cancer 8: 203-213, 2017). Additionally, P53 mutationsupregulate NRP2 expression via suppression of DLX2 transcription leadingto increased cell mobility. About 50% of human tumors and cancerscontain a mutation in the p53 tumor suppressor gene, with the vastmajority of these mutations occurring in the DNA binding domain, andsuch is linked to poorer prognosis (see, for example, Drabkin et al.,Oncotarget. 8 (No 57) 96464-96465, 2017). Additionally, TGF-β signalingis involved in the expression of NRP2b, and up-regulation of EMT incancer, which may explain why in advanced tumors, higher production ofTGF-β is positively associated with tumor aggressiveness and poorprognosis (see, for example, Malfettone et al., Cancer Lett. 392:39-50,2017).

Certain embodiments thus include methods of treating ameliorating thesymptoms of, or inhibiting the progression of, a cancer in a subject inneed thereof, comprising administering to the subject at least oneantibody or antigen-binding fragment thereof that specifically binds toa human NRP2 polypeptide (an anti-NRP2 antibody), and which modulates(e.g., interferes with) binding of the human NRP2 polypeptide to a NRP2ligand (for example, an NRP2 ligand from Table N2 or Table N3 and/or ahuman HRS polypeptide from Table H1). Certain embodiments includereducing or preventing the re-emergence of a cancer in a subject in needthereof, for example, a metastatic cancer and/or a chemoresistantcancer, wherein administration of the therapeutic composition enablesgeneration of an immune memory to the cancer.

For the treatment of cancer, in some instances, an anti-NRP2 antibodyenhances the immune response to the cancer by about, or at least about,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000% or more, relative to anuntreated control. Exemplary immune responses include increasing orenhancing immune cell invasion of a solid tumor, and increasing thebiological activity against the cancer. In certain embodiments, ananti-NRP2 antibody enhances an adaptive immune response to the cancer,and in some embodiments, an anti-NRP2 antibody enhances an innate immuneresponse to the cancer. In some-instances, an anti-NRP2 antibodydirectly or indirectly enhances a T-cell-mediated response to thecancer. In some-instances, an anti-NRP2 antibody enhances aB-cell-mediated or antibody-mediated response to the cancer. Insome-instances, an anti-NRP2 antibody modulates a macrophage responsesto the cancer. In some-instances, an anti-NRP2 antibody modulates immunecell, or cancer autophagy. In some-instances, an anti-NRP2 antibodymodulates immune cell phagocytosis. In some-instances, an anti-NRP2antibody modulates cancer cell apoptosis. In some-instances, ananti-NRP2 antibody modulates immune cell efferocytosis and/or cancercell autophagy.

In some embodiments, an anti-NRP2 antibody enhances macrophage responsesto the cancer. In some embodiments, an anti-NRP2 inhibits macrophageresponses to the cancer. In some embodiments of the anti-NRP2 antibody,the antibody enhances autophagy. In some embodiments, an anti-NRP2inhibits autophagy. In some embodiments, an anti-NRP2 enhancesphagocytosis. In some embodiments, an anti-NRP2 inhibits phagocytosis.In some embodiments, an anti-NRP2 enhances apoptosis. In someembodiments of the anti-NRP2 antibody, the antibody inhibits apoptosis.In some embodiments, an anti-NRP2 antibody enhances efferocytosis. Insome embodiments, an anti-NRP2 inhibits efferocytosis.

In some-instances, an anti-NRP2 antibody reduces cancer initiation,cancer cell migration, adhesion, or cancer cell metastasis by about orat least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or morerelative to an untreated control. In some-instances, an anti-NRP2antibody reduces cancer mediated lymphangiogenesis by about or at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative to anuntreated control.

In some embodiments, the at least one anti-NRP2 antibody reduces therate of in vitro growth of the cancer (for example, cancer cellsisolated from a biopsy or other sample grown in vitro) by about or atleast about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative toan untreated control.

In some embodiments, the at least one anti-NRP2 antibody reduces theadhesiveness of the cancer (for example, cancer cells isolated from abiopsy or other sample grown in vitro) to a substrate by about or atleast about, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative toan untreated control. In some instances, the substrate compriseslaminin.

In some embodiments, the at least one anti-NRP2 antibody reduces theinvasiveness of the cancer (for example, cancer cells isolated from abiopsy or other sample grown in vitro) by about or at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000% or more relative to an untreatedcontrol.

In some embodiments, the at least one anti-NRP2 antibody inhibits therate of migration or motility of the cancer or a migratory cell (forexample, cancer or immune cells isolated from a biopsy or other samplegrown in vitro) by about or at least about 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000% or more relative to an untreated control.

In some embodiments, the at least one anti-NRP2 antibody inhibits therate of autophagy or endosome maturation (for example, endosomeacidification) of the cancer or associated immune cells by about or atleast about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative toan untreated control.

In some embodiments, the at least one anti-NRP2 antibody enhances thesusceptibility of the cancer to an additional agent (for example,chemotherapeutic agent, hormonal therapeutic agent, and or kinaseinhibitor) by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2000% or more relative to the additional agent alone. In someembodiments, the at least one anti-NRP2 antibody enhances an anti-tumorand/or immunostimulatory activity of a cancer immunotherapy agent byabout or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% ormore, relative to the cancer immunotherapy agent alone.

Also include are combination therapies for treating cancers, includingmethods of treating ameliorating the symptoms of, or inhibiting theprogression of, a cancer in a subject in need thereof, comprisingadministering to the subject at least one antibody or antigen-bindingfragment thereof that specifically binds to a human NRP2 polypeptide (ananti-NRP2 antibody) in combination with at least one additional agent,for example, a cancer immunotherapy agent, a chemotherapeutic agent, ahormonal therapeutic agent, and/or a kinase inhibitor. Exemplary cancerimmunotherapy agents, chemotherapeutic agents, hormonal therapeuticagents, and kinase inhibitors are described elsewhere herein.

In some instances, an anti-NRP2 antibody and the at least one additionalagent are administered separately, for example, in separate therapeuticcompositions and at the same or different times. In some embodiments, ananti-NRP2 antibody and the at least one additional agent areadministered as part of the same therapeutic composition, at the sametime.

Particular methods employ one or more anti-NRP2 antibodies, orantigen-binding fragments thereof, as part of (i.e., in addition to) acombination therapy regimen. Exemplary combination regiments areprovided in Table M1 below.

TABLE M1 Combination Therapy Regimens Cancer Type Agents Acronym BreastCancer Cyclophosphamide, methotrexate, CMF 5-fluorouracil, vinorelbineDoxorubicin, cyclophosphamide AC Hodgkin's Docetaxel, doxorubicin,cyclophosphamide TAC lymphoma Doxorubicin, bleomycin, vinblastine,dacarbazine ABVD Mustine, vincristine, procarbazine, prednisolone MOPPNon-Hodgkin's Cyclophosphamide, doxorubicin, vincristine, CHOP lymphomaprednisolone Germ cell tumor Bleomycin, etoposide, cisplatin BEP Stomachcancer Epirubicin, cisplatin, 5-fluorouracil ECF Epirubicin, cisplatin,capecitabine ECX Bladder cancer Methotrexate, vincristine, doxorubicin,cisplatin MVAC Lung cancer Cyclophosphamide, doxorubicin, vincristine,CAV vinorelbine Colorectal cancer 5-fluorouracil, folinic acid,oxaliplatin FOLFOX Pancreatic Cancer Leucovorin, fluorouracil,irinotecan (Camptosar), FOLFIRINOX oxaliplatin Gemcitabine,nabpaclitaxel ABRAXANE

In some embodiments, the methods and therapeutic compositions describedherein (for example, anti-NRP2 antibody, alone or in combination with atleast one additional agent) increase median survival time of a subjectby 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or longer. In certainembodiments, the methods and therapeutic compositions described herein(for example, anti-NRP2 antibody, alone or in combination with at leastone additional agent) increase median survival time of a subject by 1year, 2 years, 3 years, or longer. In some embodiments, the methods andtherapeutic compositions described herein (for example, anti-NRP2antibody, alone or in combination with cancer immunotherapy agent)increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or longer. Incertain embodiments, the methods or therapeutic compositions describedherein increase progression-free survival by 1 year, 2 years, 3 years,or longer.

In certain embodiments, the methods and therapeutic compositionsdescribed herein (for example, anti-NRP2 antibody, alone or incombination with at least one additional agent) are sufficient to resultin tumor regression, as indicated by a statistically significantdecrease in the amount of viable tumor, for example, at least a 10%,20%, 30%, 40%, 50% or greater decrease in tumor mass, or by altered(e.g., decreased with statistical significance) scan dimensions. Incertain embodiments, the methods and therapeutic compositions describedherein (for example, anti-NRP2 antibody, alone or in combination with atleast one additional agent) are sufficient to result in stable disease.In certain embodiments, the methods and therapeutic compositionsdescribed herein (for example, anti-NRP2 antibody, alone or incombination with cancer immunotherapy agent) are sufficient to result inclinically relevant reduction in symptoms of a particular diseaseindication known to the skilled clinician.

In some embodiments, an anti-NRP2 antibody increases, complements, orotherwise enhances the anti-tumor and/or immunostimulatory activity ofthe cancer immunotherapy agent, relative to the cancer immunotherapyagent alone. In some embodiments, an anti-NRP2 antibody enhances theanti-tumor and/or immunostimulatory activity of the cancer immunotherapyagent by about, or at least about, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,2000% or more, relative to the cancer immunotherapy agent alone.

The methods and therapeutic compositions described herein can be used inthe treatment of any variety of cancers or tumors. In some embodiments,the cancer is a primary cancer, i.e., a cancer growing at the anatomicalsite where tumor progression began and yielded a cancerous mass. In someembodiments, the cancer is a secondary or metastatic cancer, i.e., acancer which has spread from the primary site or tissue of origin intoone or more different sites or tissues. In some embodiments, the cancerexpresses or overexpresses NRP2. In some embodiments, the subject orpatient has a cancer selected from one or more of melanoma (e.g.,metastatic melanoma), an epithelial or epithelial-derived tumor,pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer,non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g.,lymphocytic leukemia, chronic myelogenous leukemia, acute myeloidleukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma(hepatocellular carcinoma or HCC), sarcoma, B-cell malignancy, breastcancer (for example, estrogen receptor positive (ER+), estrogen receptornegative (ER−), Her2 positive (Her2+), Her2 negative (Her2−), or acombination thereof, e.g., ER+/Her2+, ER+/Her2−, ER−/Her2+, orER−/Her2−; or “triple negative” breast cancer which is estrogenreceptor-negative, progesterone receptor-negative, and HER2-negative),ovarian cancer, colorectal cancer, glioma (e.g., astrocytoma,oligodendroglioma, ependymoma, or a choroid plexus papilloma),glioblastoma multiforme (e.g., giant cell gliobastoma or a gliosarcoma),meningioma, pituitary adenoma, vestibular schwannoma, primary CNSlymphoma, primitive neuroectodermal tumor (medulloblastoma), kidneycancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer,esophageal cancer, brain cancer, head and neck cancers, cervical cancer,testicular cancer, thyroid cancer, stomach cancer, virus-induced tumorssuch as, for example, papilloma virus-induced carcinomas (e.g., cervicalcarcinoma, cervical cancer), adenocarcinomas, herpes virus-inducedtumors (e.g., Burkitt's lymphoma, EBV-induced B-cell lymphoma),hepatitis B-induced tumors (hepatocellular carcinomas), HTLV-1-induedand HTLV-2-induced lymphomas, acoustic neuroma, lung cancers (e.g., lungcarcinoma, bronchial carcinoma), small-cell lung carcinomas, pharyngealcancer, anal carcinoma, glioblastoma, rectal carcinoma, lymphangioma,astrocytoma, brain tumors, retinoblastoma, basalioma, brain metastases,medulloblastomas, vaginal cancer, pancreatic cancer, testicular cancer,Hodgkin's syndrome, meningiomas, Schneeberger disease, hypophysis tumor,Mycosis fungoides, carcinoids, neurinoma, spinalioma, Burkitt'slymphoma, laryngeal cancer, renal cancer, thymoma, corpus carcinoma,bone cancer, non-Hodgkin's lymphomas, urethral cancer, CUP syndrome,head/neck tumors, oligodendroglioma, vulval cancer, intestinal cancer,colon carcinoma, oesophageal cancer (e.g., oesophageal carcinoma), wartinvolvement, tumors of the small intestine, craniopharyngeomas, ovariancarcinoma, genital tumors, ovarian cancer (e.g., ovarian carcinoma),pancreatic cancer (e.g., pancreatic carcinoma), endometrial carcinoma,liver metastases, penile cancer, tongue cancer, gall bladder cancer,leukaemia, plasmocytoma, and lid tumor.

In some embodiments, as noted above, the cancer or tumor is a metastaticcancer, for example, a metastatic cancer that expresses NRP2a and/orNRP2b. Further to the above cancers, exemplary metastatic cancersinclude, without limitation, bladder cancers which have metastasized tothe bone, liver, and/or lungs; breast cancers which have metastasized tothe bone, brain, liver, and/or lungs; colorectal cancers which havemetastasized to the liver, lungs, and/or peritoneum; kidney cancerswhich have metastasized to the adrenal glands, bone, brain, liver,and/or lungs; lung cancers which have metastasized to the adrenalglands, bone, brain, liver, and/or other lung sites; melanomas whichhave metastasized to the bone, brain, liver, lung, and/or skin/muscle;ovarian cancers which have metastasized to the liver, lung, and/orperitoneum; pancreatic cancers which have metastasized to the liver,lung, and/or peritoneum; prostate cancers which have metastasized to theadrenal glands, bone, liver, and/or lungs; stomach cancers which havemetastasized to the liver, lung, and/or peritoneum; thyroid cancerswhich have metastasized to the bone, liver, and/or lungs; and uterinecancers which have metastasized to the bone, liver, lung, peritoneum,and/or vagina; among others.

In some embodiments, for example, where the cancer immunotherapy agentis a PD-1 or PD-L1 antagonist or inhibitor, the subject has one or morebiomarkers (e.g., increased PD-1 or PD-L1 levels in cells such as cancercells or cancer-specific CTLs) that make the suitable for PD-1 or PD-L1inhibitor therapy. For instance, in some embodiments, the subject hasincreased fractions of programmed cell death 1 high/cytotoxic Tlymphocyte-associated protein 4 high (e.g., PD-1^(hi)CTLA-4^(hi)) cellswithin a tumor-infiltrating CD8+ T cell subset (see, e.g., Daud et al.,J Clin Invest. 126:3447-3452, 2016). As another example, in someembodiments, the subject has increased levels of Bim (B cell lymphoma2-interacting (Bcl2-interacting) mediator) in circulating tumor-reactive(e.g., PD-1⁺CD11a^(hi)CD8⁺) T cells, and optionally has metastaticmelanoma (see, e.g., Dronca et al., JCI Insight. May 5; 1(6): e86014,2016).

Certain specific combinations include an anti-NRP2 antibody and a PD-L1antagonist or inhibitor, for example, atezolizumab (MPDL3280A), avelumab(MSB0010718C), and durvalumab (MEDI4736), for treating a cancer selectedfrom one or more of colorectal cancer, melanoma, breast cancer,non-small-cell lung carcinoma, bladder cancer, and renal cell carcinoma.

Some specific combinations include an anti-NRP2 antibody and a PD-1antagonist, for example, nivolumab, for treating a cancer selected fromone or more of Hodgkin's lymphoma, melanoma, non-small cell lung cancer,hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer.

Particular specific combinations include an anti-NRP2 antibody and aPD-1 antagonist, for example, pembrolizumab, for treating a cancerselected from one or more of melanoma, non-small cell lung cancer, smallcell lung cancer, head and neck cancer, and urothelial cancer.

Certain specific combinations include an anti-NRP2 antibody and a CTLA-4antagonist, for example, ipilimumab and tremelimumab, for treating acancer selected from one or more of melanoma, prostate cancer, lungcancer, and bladder cancer.

Some specific combinations include an anti-NRP2 antibody and an IDOantagonist, for example, indoximod (NLG-8189), 1-methyl-tryptophan(1MT), β-Carboline (norharmane; 9H-pyrido[3,4-b]indole), rosmarinicacid, or epacadostat, for treating a cancer selected from one or more ofmetastatic breast cancer and brain cancer optionally GlioblastomaMultiforme, glioma, gliosarcoma or malignant brain tumor.

Certain specific combinations include an anti-NRP2 antibody and thecytokine INF-α for treating melanoma, Kaposi sarcoma, and hematologiccancers. Also included is the combination of an anti-NRP2 antibody andIL-2 (e.g., Aldesleukin) for treating metastatic kidney cancer ormetastatic melanoma.

Some specific combinations include an anti-NRP2 antibody and a T-cellbased adoptive immunotherapy, for example, comprising CAR-modifiedT-cells targeted against CD-19, for treating hematological cancers suchas acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia(CLL), and B-cell neoplasms (see, e.g., Maude et al., 2015, supra;Lorentzen and Straten, Scand J Immunol. 82:307-19, 2015; and Ramos etal., Cancer J. 20:112-118, 2014).

The methods for treating cancers can be combined with other therapeuticmodalities. For example, a combination therapy described herein can beadministered to a subject before, during, or after other therapeuticinterventions, including symptomatic care, radiotherapy, surgery,transplantation, hormone therapy, photodynamic therapy, antibiotictherapy, or any combination thereof. Symptomatic care includesadministration of corticosteroids, to reduce cerebral edema, headaches,cognitive dysfunction, and emesis, and administration ofanti-convulsants, to reduce seizures. Radiotherapy includes whole-brainirradiation, fractionated radiotherapy, and radiosurgery, such asstereotactic radiosurgery, which can be further combined withtraditional surgery.

Certain embodiments include the use of the anti-NRP2 antibodiesdescribed herein to modulate lymphangiogenesis, and treat relatedconditions such as lymphedema or tumor metastasis. The lymphatic systemconsists of networks of interconnected capillaries, collecting vesselsand lymph nodes that absorb, collect and transport the fluid and proteinfiltered from the blood vascular system. This system provides a criticalhomeostatic function: in humans, lymphatic vessels return >4 liters offluid and a substantial amount of protein per day back into the greatveins of the neck.

Lymphatic vascular dysfunction (lymphedema) results in the accumulationof excess fluid (edema) in the interstitium. Although lymphedema istypically not life-threatening, it has serious health consequences,including pain, immobility, fibrosis, inflammation, adipose tissueaccumulation, and tissue damage. Because the lymphatic system is also acritical component of immune responses, lymphedema is typicallyaccompanied by an increased risk of infection and other immune systemproblems.

Lymphangiogenesis is the formation of new lymphatic vessels frompreexisting lymphatic vessels and is associated with diversepathological conditions including metastatic dissemination, graftrejection (e.g., cornea, kidney and heart), type 2 diabetes, obesity,hypertension, and lymphedema (See, e.g. Alitalo et al. Nature438:946-953, 2005; Karaman et al. J Clin Invest 124:922-928, 2014; Kimet al., J Clin Invest 124:936-942, 2014; Maby-El Hajjami et al.,Histochem Cell Biol 130:1063-107, 2008; Machnik et al., Nat Med15:545-552; Mortimer et al., 2014. J Clin Invest 124:915-921; Skobe etal., 2009. Nat Med 15:993-994).

Lymphatic vessel invasion in and around a primary tumor compared toinvasion of blood vessels is a prognostic marker of the aggressivenessof various types of cancers. Growth of lymphatic vessels is alsoinvolved in graft rejection (Dietrich, T., et al., J Immunol184:535-539, 2010, Hall et al., Arch Otolaryngol Head Neck Surg129:716-719, 2003; Maula et al., Cancer Res 63:1920-1926, 2003; Miyataet al., J Urol 176:348-353, 2006; Saad et al., Mod Pathol 19:1317-1323,2006; Schoppmann et al., Ann Surg 240:306-312, 2004; Zeng et al.,Prostate 65:222-230, 2005).

Despite the well-established significance of lymphatics in thepathogenesis of numerous diseases, there has been little progress in thedevelopment of anti-lymphangiogenic agents compared to the abundance ofanti-angiogenic agents that have entered clinical trials. Therefore, thedevelopment of additional lymphangiogenesis inhibitors is of interestfor the treatment of a range of conditions, including lymphedema andcancer metastasis.

Anti-lymphangiogenic agents are useful, for example, for treatment ofdebilitating diseases of the eye, where the growth of lymphatic vesselsis the major reason of corneal graft rejection and a major contributorto neovascularization associated with age related macular degeneration(Dietrich, T., et al., J Immunol 184:535-539, 2010). In particular,penetrating keratoplasty is the most common form of solid tissuetransplantation, with approximately 40,000 corneal transplantationsperformed each year in the United States. The success rate ofpenetrating keratoplasty is as high as 90% for uncomplicated firstgrafts performed in avascular low-risk beds. However, the rejection rateof the corneal grafts placed in high-risk vascularized host beds isextremely high (70% to 90%). Thus the development of safe and targetednew regimens to inhibit lymphangiogenesis are needed to promote graftsurvival and inhibit neovascularization

Anti-lymphangiogenesis drugs are useful also for treatment of dry eyedisease. Significant upregulation of pro-lymphangiogenic factors (e.g.,VEGF-C, VEGF-D, and VEGFR-3) and selective growth of lymphatic vesselswithout concurrent growth of blood vessels has been demonstrated incorneas with dry eye disease (Goyal et al., Arch Ophthalmol 128:819-824,2010). Dry eye disease is an immune-mediated disorder affecting about 5million Americans. It severely impacts the vision-related quality oflife and the symptoms can be debilitating. The current therapeuticoptions for dry eye disease are limited, mostly palliative, andexpensive. Therefore, development of lymphangiogenesis inhibitors is oftherapeutic value for treatment of dry eye disease.

It is currently accepted that metastases are responsible for the vastmajority (90%) of deaths from solid tumors (Gupta and Massague, Cell127, 679-695, 2006). The complex process of metastasis involves a seriesof distinct steps including detachment of tumor cells from the primarytumor, intravasation of tumor cells into lymphatic or blood vessels, andextravasation and growth of tumor cells in secondary sites. Analysis ofregional lymph nodes in many tumor types suggests that the lymphaticvasculature is an important route for the dissemination of humancancers. Furthermore, in almost all carcinomas, the presence of tumorcells in lymph nodes is the most important adverse prognostic factor.While it was previously thought that such metastases exclusivelyinvolved passage of malignant cells along pre-existing lymphatic vesselsnear tumors, recent experimental studies and clinicopathological reports(See, e.g., Achen et al., Br J Cancer 94, 1355-1360, 2006 and Nathanson,Cancer 98, 413-423, 2003) suggest that lymphangiogenesis can be inducedby solid tumors and can promote tumor spread. These and other recentstudies suggest targeting lymphatics and lymphangiogenesis may be auseful therapeutic strategy to restrict the development of cancermetastasis, which would have a significant benefit for many patients.

Accordingly, there is a need for methods and compositions that inhibitthe activities of pro-lymphangiogenic factors and for methods thatprevent or treat graft rejection, dry-eye disease tumor metastasis,lymphedema, and inflammatory conditions.

Given the role of NRP2 in these processes, anti-NRP2 antibodies orantigen-binding fragments thereof represent powerful new tools for thedevelopment of both pro and anti-lymphangiogenic therapies. Suchdifferential effects may be mediated for example, via the uses ofdifferent anti-NRP2 compositions, differential NRP2a or NRP2bselectivity, differential dosing, differential treatment duration, orthe appropriate use of additional co-factors, such as, for example,VEGF-C, or Semaphorin 3F and/or 3G.

Accordingly, in some embodiments, the at least one antibody orantigen-binding fragment thereof selectively modulates the bindingand/or signaling of VEGF-C or related NRP2 ligands to or via an NRP2polypeptide. In some aspects, such antibodies or antigen-bindingfragment thereof do not substantially block the interaction ofsemaphorins to an NRP2 polypeptide. In some embodiments, the at leastone antibody or antigen-binding fragment thereof selectively modulatesthe binding and/or signaling of Semaphorin 3F or related NRP2 ligands toor via an NRP2 polypeptide. In some aspects, such antibodies do notsubstantially block the interaction of VEGF-C to an NRP2 polypeptide. Insome embodiments, such antibodies selectively modulate both the bindingof VEGF-C or related NRP2 ligands and semaphorins to an NRP2polypeptide.

In some embodiments, such antibodies are agonistic antibodies withrespect to VEGF-C signaling. In some aspects, such antibodies areantagonistic antibodies with respect to VEGF-C signaling. In someembodiments, such antibodies are agonistic antibodies with respect tosemaphorin 3F signaling. In some aspects, such antibodies areantagonistic antibodies with respect to semaphorin 3F signaling.

In some embodiments, the at least one antibody or antigen-bindingfragment thereof antagonizes the binding/signaling activity between anNRP2 polypeptide and VEGFR2 or VEGFR3 without substantially modulatingthe binding/signaling activity between the NRP2 polypeptide and a plexinreceptor and/or a semaphorin. In some embodiments, the at least oneantibody or antigen-binding fragment thereof antagonizes thebinding/signaling activity between an NRP2 polypeptide and a plexinreceptor and/or a semaphorin without substantially modulating thebinding/signaling activity between the NRP2 polypeptide and VEGFR2,VEGFR3, and/or VEGF-C.

Certain embodiments thus include methods of treating ameliorating thesymptoms of, or inhibiting the progression of, lymphangiogenesis, orneovascularization in a subject in need thereof, comprisingadministering to the subject at least one antibody or antigen-bindingfragment thereof that specifically binds to a human NRP2 polypeptide,and which modulates (e.g., interferes with) binding of the human NRP2polypeptide to VEGF-C, VEGFR1, VEGFR2, and/or VEGFR3.

Some embodiments include the use of the anti-NRP2 antibodies describedherein to modulate smooth muscle contractility, and treat relatedconditions. Reduced smooth muscle (SM) contractility in the bladder canstem from numerous etiologies including partial obstruction secondary tobenign prostatic hyperplasia (BPH), posterior urethral valves, diabetesmellitus, multiple sclerosis, spinal cord injury, or idiopathic causes.(See, e.g., Drake et al., Nat Rev Urol. 11(8):454-464, 2014). Inconditions such as BPH or posterior urethral valves, the bladdercontracts against an obstructed outlet. The initial response isadaptive, involving a compensatory phase of SM hypertrophy that enablesincreased force generation to overcome the increased outlet resistance.When the demand outstrips the adaptive capability of the bladder,contractile performance becomes less efficient, residual volumesincrease, and the bladder remodels, ultimately leading to a loss ofdetrusor contractility as the bladder decompensates. (See, e.g., Zdericet al., J Cell Mol Med. 16(2):203-217, 2012). The prevalence ofunderactive detrusor function is reported to be as high as 48% in adults(Osman et al., Eur Urol; 65(2):389-398, 2014). Furthermore, existingpharmacological treatments for restoration of SM contraction such asmuscarinic agonists or cholinesterase inhibitors have shown limitedefficacy and adverse effects (Barendrecht et al., BJU Int.99(4):749-752, 2007).

Recent studies have identified bladder smooth muscle as a major site ofNrp2 expression, demonstrated the inhibition of RhoA and cytoskeletalstiffness, and observed increased contractility of bladder SM stripsfrom mice with ubiquitous or smooth muscle-specific deletion of NRP2 invivo, when compared with tissues from NRP2-intact littermate controls(See, e.g., Bielenberg et al., Am. J. Pathol. 181548-559, 2012; andVasquez et al., JCI Insight 2(3) e90617, 2017).

Collectively, these findings suggest that downregulation of NRP2 is animportant component of the compensatory response to obstruction inexperimental animals and in humans with obstruction, and suggest thatNrp2 may represent a novel pharmacological target for maintenance orrestoration of detrusor contractility in the decompensated bladder.

Furthermore, recent studies have shown that targeting NRP2 in bladdersundergoing decompensation has the potential to restore contractility inspite of ongoing obstruction. (Vasquez et al., JCI Insight 2(3) e90617,2017). These findings argue that the NRP2 axis represents a potentiallynovel pharmacologic target for restoration of SM contractility inpartial bladder outlet obstruction syndromes, and provide an importanttherapeutic opportunity for the development of antibody-based modulatorsof NRP2 function.

Pharmacological management of diminished detrusor contractility to datehas focused on stimulation of parasympathetic activity to enhancebladder contractility and reduction of outflow resistance to facilitatebladder emptying (Chancellor et al., Urology 72(5) 966-967, 2008).However, analysis of 10 randomized clinical trials ofparasympathomimetic drugs in patients with poorly contractile bladders,revealed either a worsening of symptoms or a lack of significantimprovement (Barendrecht et al., BJU Int. 99(4) 749-752, 2007). Theincrease in contractility following NRP2 deletion in the decompensatingbladder suggests that NRP2 may be a useful target to mitigate reduceddetrusor contractility under conditions of chronic obstruction.

Given the role of NRP2 in this process, the anti-NRP2 antibodiesdescribed herein could provide new therapies to modulate smooth musclecontractility, including for example, the treatment of reduced smoothmuscle (SM) contractility in the bladder, and more specificallysyndromes associated with partial bladder outlet obstruction syndromes.

Certain embodiments thus include methods of treating ameliorating thesymptoms of, or inhibiting the progression of partial bladder outletobstruction syndromes in a subject in need thereof, comprisingadministering to the subject at least one antibody or antigen-bindingfragment thereof that specifically binds to a human NRP2 polypeptide (ananti-NRP2 antibody), and which modulates (e.g., interferes with) bindingof the human NRP2 polypeptide to a NRP2 ligand (for example, an NRP2ligand from Table N2 or Table N3).

Certain embodiments also include methods of modulating (e.g.,increasing, reducing) smooth muscle contractility in a subject in needthereof, comprising administering to the subject an anti-NRP2 antibody,or antigen-binding fragment thereof, or therapeutic composition providedherein. Certain embodiments include treating, ameliorating the symptomsof, and/or reducing the progression of, reduced smooth musclecontractility in a subject in need thereof, comprising administering tothe subject an anti-NRP2 antibody or antigen-binding fragment thereof,or therapeutic composition provided herein.

Methods for identifying subjects with one or more of the diseases orconditions described herein are known in the art. In some embodiments,the subject has, and/or is selected for treatment based on having, adisease associated with increased levels or expression of at least oneNRP2 ligand (for example, an NRP2 ligand from Table N2 or Table N3and/or an HRS polypeptide from Table H1) and/or a coding mRNA thereofrelative to a healthy control. For instance, in some embodiments, thelevels of the at least one NRP2 ligand in the diseases subject, cells,or tissue are about or at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or more times the levelsof the at least one NRP2 ligand in a healthy control. In someembodiments, the subject has, and/or is selected for treatment based onhaving, a cancer which has increased levels or expression of at leastone NRP2 ligand (for example, an NRP2 ligand from Table N2 or Table N3and/or an HRS polypeptide from Table H1) and/or a coding mRNA thereofrelative to a non-cancerous control cell or tissue. For instance, insome embodiments, the levels of the at least one NRP2 ligand in thecancer cells or tissue are about or at least about 1.5, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or more timesthe levels of the NRP2 ligand in a non-cancerous control or standard.Thus, certain embodiments include methods of selecting a subject fortreatment, comprising (i) detecting increased expression levels of atleast one NRP2 ligand (for example, an NRP2 ligand from Table N2 orTable N3 and/or an HRS polypeptide from Table H1) and/or coding mRNA inthe subject relative to a control or reference, and (ii) administeringto the subject a therapeutic composition comprising at least oneanti-NRP2-antibody or antigen-binding fragment thereof, as describedherein. In particular embodiments, the HRS polypeptide is a splicevariant of full-length HRS. In some embodiments, the HRS splice variantis selected from one or more of HisRSN1, HisRSN2, HisRSN3, HisRSN4,HisRSN5, HisRSC1, HisRSC2, HisRSC3, HisRSC4, HisRSC5, HisRSC6, HisRSC7,HisRSC8, and HisRSC9.

In some embodiments, the subject has, and/or is selected for treatmentbased on having, increased circulating or serum levels of a solubleneuropilin 2 (NRP2) polypeptide (selected, for example, from Table N1),either bound to an HRS polypeptide or free, relative to the levels of ahealthy or matched control population of subject(s). For instance, incertain embodiments, the circulating or serum levels are about or atleast about 10, 20, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000,4000, 5000 pM of the soluble NRP2 polypeptide, or the circulating orserum levels are about 30-50, 50-100, 100-2000, 200-2000, 300-2000,400-2000, 500-2000, 600-2000, 700-2000, 800-2000, 900-2000, 1000-2000,2000-3000, 3000-4000, 4000-5000 pM of the soluble NRP2 polypeptide.

In certain embodiments, the subject has, and/or is selected fortreatment based on having, a disease associated with increased levels orexpression of an NRP2 polypeptide (optionally selected from Table N1)and/or a coding mRNA thereof relative to a healthy control (e.g., anNRP2-associated disease). In some embodiments, the NRP2 polypeptide isan NRP2a isoform (e.g., variants 1, 2, and/or 3 of Table N1) or afragment thereof. In some embodiments, the NRP2 polypeptide is an NRP2bisoform (e.g., variants 4 and/or 5 of Table N1) or a fragment thereof.

For example, in certain embodiments, the levels of the NRP2 polypeptidein the diseased subject, cells, or tissue are about or at least about1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100or more times the levels of NRP2 polypeptide in a healthy control. Insome embodiments, the subject has, and/or is selected for treatmentbased on having, a cancer which has increased levels or expression of aNRP2 polypeptide (selected, for example, from Table N1) and/or a codingmRNA thereof relative to a control cell or tissue, optionally relativeto a non-cancerous cell or tissue of the same type as the cancer. Forinstance, in some embodiments, the levels of the NRP2 polypeptide in thecancer cells or tissue are about or at least about 1.5, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more times thelevels of NRP2 polypeptide in a non-cancerous control or standard. Someembodiments thus include methods of selecting a subject for treatment,comprising (i) detecting increased expression levels of a NRP2polypeptide and/or a coding mRNA thereof in the subject relative to acontrol or reference, and (ii) administering to the subject atherapeutic composition comprising at least one anti-NRP2-antibody orantigen-binding fragment thereof, as described herein.

In some embodiments, the subject has, and/or is selected for treatmentbased on having, a disease associated with increased levels orexpression of NRP2a (e.g., variants 1, 2, and/or 3 of Table N1) and/orNRP2b (e.g., variants 4 and/or 5 of Table N1), or an altered ratio ofNRP2a:NRP2b expression, relative to a healthy control or matched controlstandard or population of subject(s). In some embodiments, the subjecthas significantly higher expression or levels of NRP2b relative to ahealthy control or matched control standard or population of subject(s).In some embodiments, the levels of NRP2b are increased by about or atleast about 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, 600%,700%, 800%, 900%, 1000% compared to a healthy control or matched controlstandard or population of subject(s). In some embodiments, the subjecthas, and/or is selected for treatment based on having, increasedcirculating levels of HRS:NRP2 complexes relative to a healthy ormatched control standard or population of subject(s). Certainembodiments therefore include methods of selecting a subject for cancertreatment, comprising (i) detecting increased expression levels ofHRS:NRP2 complexes in the subject relative to a control or reference,and (ii) administering to the subject a therapeutic compositioncomprising at least one anti-NRP2-antibody or antigen-binding fragmentthereof, as described herein.

In some embodiments, the healthy control or matched control standard orpopulation of subject(s) comprises average ranges for age-matchedsamples of diseased or non-diseased cells or tissue of the same type,which comprise specific characteristics such as drug resistance,metastatic potential, aggressiveness, genetic signature (e.g., p53mutations, PTEN deletion, IGFR expression), and/or expression patterns.

For in vivo use, as noted above, for the treatment of human or non-humanmammalian disease or testing, the agents described herein are generallyincorporated into one or more therapeutic or pharmaceutical compositionsprior to administration, including veterinary therapeutic compositions.

Thus, certain embodiments relate to therapeutic compositions thatcomprise at least one antibody or antigen-binding fragment thereof thatspecifically binds to a human NRP2 polypeptide, as described herein. Insome instances, a therapeutic or pharmaceutical composition comprisesone or more of the agents described herein in combination with apharmaceutically- or physiologically-acceptable carrier or excipient.Certain therapeutic compositions further comprise at least one cancerimmunotherapy agent, as described herein.

Some therapeutic compositions comprise (and certain methods utilize)only one anti-NRP2 antibody or antigen-binding fragment thereof. Certaintherapeutic compositions comprise (and certain methods utilize) amixture of at least two, three, four, or five different anti-NRP2antibodies or antigen-binding fragments thereof.

For instance, certain therapeutic compositions comprise at least twoanti-NRP2 antibodies, including a first antibody or antigen-bindingfragment thereof that specifically binds to at least one first epitopeof a human NRP2 polypeptide, and a second antibody or antigen-bindingfragment thereof that specifically binds to at least one second epitopeof a human NRP2 polypeptide, wherein the at least one first epitopediffers from the at least one second epitope. In some embodiments, thefirst and the second antibody or antigen-binding fragment thereofspecifically and non-competitively bind to the same domain of the NRP2polypeptide. In some embodiments, the first anti-NRP2 antibody orantigen-binding fragment thereof binds selectively to a first epitopethat is specific to the NRP2a isoform (e.g., variants 1, 2, and/or 3 ofTable N1), and the second anti-NRP2 antibody or antigen-binding fragmentthereof binds selectively to a second epitope which is specific to theNRP2b isoform (e.g., variants 4 and/or 5 of Table N1).

In some embodiments, the first and the second antibody orantigen-binding fragment thereof specifically and non-competitively bindto different domains of the NRP2 polypeptide.

In some embodiments, the first antibody antagonizes thebinding/signaling activity between the NRP2 polypeptide and the at leastone NRP2 ligand. In certain embodiments, the second antibody orantigen-binding fragment thereof agonizes or enhances thebinding/signaling activity between the NRP2 polypeptide and at least oneNRP2 ligand.

In some embodiments, the first and the second antibody orantigen-binding fragments thereof are both blocking antibodies, forexample, for at least two different NRP2 ligands. In some embodiments,the first and the second antibody or antigen-binding fragments thereofare both partial-blocking antibodies, for example, for at least twodifferent NRP2 ligands. In some instances, the first and the secondantibodies or antigen-binding fragments thereof are both non-blockingantibodies, for example, with respect to at least two different NRP2ligands.

In some instances, the first antibody or antigen-binding fragmentthereof is a blocking antibody and the second antibody orantigen-binding fragment thereof is a partial-blocking antibody. Incertain instances, the first antibody or antigen-binding fragmentthereof is a blocking antibody and the second antibody orantigen-binding fragment thereof is a non-blocking antibody.

In some embodiments, the first and the second antibodies orantigen-binding fragments thereof both comprise an IgG Fc domain withhigh effector function in humans, for example, an IgG1 or IgG3 Fcdomain. In some embodiments, the first and the second antibodies orantigen-binding fragments thereof comprise an IgG Fc domain with loweffector function in humans, for example, an IgG2 or IgG4 Fc domain.

In some instances, the first antibody or antigen-binding fragmentthereof comprises an IgG Fc domain with high effector function inhumans, for example, an IgG1 or IgG3 Fc domain, and the second antibodyor antigen-binding fragment thereof comprises an IgG Fc domain with loweffector function in humans, for example, an IgG2 or IgG4 Fc domain.

In particular embodiments, the therapeutic composition comprising theagents such as antibodies or other polypeptide agents (e.g., anti-NRP2antibodies) is substantially pure on a protein basis or a weight-weightbasis, for example, the composition has a purity of at least about 80%,85%, 90%, 95%, 98%, or 99% on a protein basis or a weight-weight basis.

In some embodiments, the antibodies (e.g., anti-NRP2 antibodies) orother polypeptide agents provided herein do not form aggregates, have adesired solubility, and/or have an immunogenicity profile that issuitable for use in humans, as described herein and known in the art.Thus, in some embodiments, the therapeutic composition comprising apolypeptide agent (for example, an antibody such as an anti-NRP2antibody) is substantially aggregate-free. For example, certaincompositions comprise less than about 10% (on a protein basis) highmolecular weight aggregated proteins, or less than about 5% highmolecular weight aggregated proteins, or less than about 4% highmolecular weight aggregated proteins, or less than about 3% highmolecular weight aggregated proteins, or less than about 2% highmolecular weight aggregated proteins, or less than about 1% highmolecular weight aggregated proteins. Some compositions comprise apolypeptide agent (e.g., an antibody such as an anti-NRP2 antibody) thatis at least about 50%, about 60%, about 70%, about 80%, about 90% orabout 95% monodisperse with respect to its apparent molecular mass.

In some embodiments, polypeptide agents such as antibodies (e.g.,anti-NRP2 antibodies) are concentrated to about or at least about 0.1mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6, 0.7, 0.8, 0.9, 1mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9mg/ml, 10 mg/ml, 11, 12, 13, 14 or 15 mg/ml and are formulated forbiotherapeutic uses.

To prepare a therapeutic or pharmaceutical composition, an effective ordesired amount of one or more agents is mixed with any pharmaceuticalcarrier(s) or excipient known to those skilled in the art to be suitablefor the particular agent and/or mode of administration. A pharmaceuticalcarrier may be liquid, semi-liquid or solid. Solutions or suspensionsused for parenteral, intradermal, intraocular, subcutaneous, directinstillation into the bladder, or topical application may include, forexample, a sterile diluent (such as water), saline solution (e.g.,phosphate buffered saline; PBS), fixed oil, polyethylene glycol,glycerin, propylene glycol or other synthetic solvent; antimicrobialagents (such as benzyl alcohol and methyl parabens); antioxidants (suchas ascorbic acid and sodium bisulfite) and chelating agents (such asethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates,citrates and phosphates). If administered intravenously (e.g., by IVinfusion), suitable carriers include physiological saline or phosphatebuffered saline (PBS), and solutions containing thickening andsolubilizing agents, such as glucose, polyethylene glycol, polypropyleneglycol and mixtures thereof.

Administration of agents described herein, in pure form or in anappropriate therapeutic or pharmaceutical composition, can be carriedout via any of the accepted modes of administration of agents forserving similar utilities. The therapeutic or pharmaceuticalcompositions can be prepared by combining an agent-containingcomposition with an appropriate physiologically acceptable carrier,diluent or excipient, and may be formulated into preparations in solid,semi-solid, liquid or gaseous forms, such as tablets, capsules, powders,granules, ointments, solutions, suppositories, injections, inhalants,gels, microspheres, and aerosols. In addition, other pharmaceuticallyactive ingredients (including other small molecules as describedelsewhere herein) and/or suitable excipients such as salts, buffers andstabilizers may, but need not, be present within the composition.

Administration may be achieved by a variety of different routes,including oral, parenteral, nasal, intravenous, intraocular,intradermal, intramuscular, subcutaneous, installation into the bladder,or topical. Preferred modes of administration depend upon the nature ofthe condition to be treated or prevented. Particular embodiments includeadministration by IV infusion.

Carriers can include, for example, pharmaceutically- orphysiologically-acceptable carriers, excipients, or stabilizers that arenon-toxic to the cell or mammal being exposed thereto at the dosages andconcentrations employed. Often the physiologically-acceptable carrier isan aqueous pH buffered solution. Examples of physiologically acceptablecarriers include buffers such as phosphate, citrate, and other organicacids; antioxidants including ascorbic acid; low molecular weight (lessthan about 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as polysorbate 20 (TWEEN™) polyethylene glycol (PEG), andpoloxamers (PLURONICS™), and the like.

In some embodiments, one or more agents can be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate)microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980). The particle(s) or liposomes may further comprise othertherapeutic or diagnostic agents.

The precise dosage and duration of treatment is a function of thedisease being treated and may be determined empirically using knowntesting protocols or by testing the compositions in model systems knownin the art and extrapolating therefrom. Controlled clinical trials mayalso be performed. Dosages may also vary with the severity of thecondition to be alleviated. A pharmaceutical composition is generallyformulated and administered to exert a therapeutically useful effectwhile minimizing undesirable side effects. The composition may beadministered one time, or may be divided into a number of smaller dosesto be administered at intervals of time. For any particular subject,specific dosage regimens may be adjusted over time according to theindividual need.

Typical routes of administering these and related therapeutic orpharmaceutical compositions thus include, without limitation, oral,topical, transdermal, inhalation, parenteral, sublingual, buccal,ocular, rectal, vaginal, and intranasal. The term parenteral as usedherein includes subcutaneous injections, intravenous, instillation intothe bladder, intramuscular, intrasternal injection or infusiontechniques. Therapeutic or pharmaceutical compositions according tocertain embodiments of the present disclosure are formulated so as toallow the active ingredients contained therein to be bioavailable uponadministration of the composition to a subject or patient. Compositionsthat will be administered to a subject or patient may take the form ofone or more dosage units, where for example, a tablet may be a singledosage unit, and a container of a herein described agent in aerosol formmay hold a plurality of dosage units. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in thisart; for example, see Remington: The Science and Practice of Pharmacy,20th Edition (Philadelphia College of Pharmacy and Science, 2000). Thecomposition to be administered will typically contain a therapeuticallyeffective amount of an agent described herein, for treatment of adisease or condition of interest.

A therapeutic or pharmaceutical composition may be in the form of asolid or liquid. In one embodiment, the carrier(s) are particulate, sothat the compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral oil, injectable liquid or an aerosol, which is useful in, forexample, inhalatory administration. When intended for oraladministration, the pharmaceutical composition is preferably in eithersolid or liquid form, where semi-solid, semi-liquid, suspension and gelforms are included within the forms considered herein as either solid orliquid. Certain embodiments include sterile, injectable solutions.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, gel, compressedtablet, pill, capsule, chewing gum, wafer or the like. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent. When the pharmaceutical composition isin the form of a capsule, for example, a gelatin capsule, it maycontain, in addition to materials of the above type, a liquid carriersuch as polyethylene glycol or oil.

The therapeutic or pharmaceutical composition may be in the form of aliquid, for example, an elixir, syrup, solution, gel, emulsion orsuspension. The liquid may be for oral administration or for delivery byinjection, as two examples. When intended for oral administration,preferred composition contain, in addition to the present compounds, oneor more of a sweetening agent, preservatives, dye/colorant and flavorenhancer. In a composition intended to be administered by injection, oneor more of a surfactant, preservative, wetting agent, dispersing agent,suspending agent, buffer, stabilizer and isotonic agent may be included.

The liquid therapeutic or pharmaceutical compositions, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid therapeutic or pharmaceutical composition intended for eitherparenteral, intraocular, or oral administration should contain an amountof an agent such that a suitable dosage will be obtained. Typically,this amount is at least 0.01% of the agent of interest in thecomposition. When intended for oral administration, this amount may bevaried to be between 0.1 and about 70% of the weight of the composition.Certain oral therapeutic or pharmaceutical compositions contain betweenabout 4% and about 75% of the agent of interest. In certain embodiments,therapeutic or pharmaceutical compositions and preparations are preparedso that a parenteral dosage unit contains between 0.01 to 10% by weightof the agent of interest prior to dilution.

The therapeutic or pharmaceutical compositions may be intended fortopical administration, in which case the carrier may suitably comprisea solution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in atherapeutic or pharmaceutical composition for topical administration. Ifintended for transdermal administration, the composition may include atransdermal patch or iontophoresis device.

The therapeutic or pharmaceutical compositions may be intended forrectal administration, in the form, for example, of a suppository, whichwill melt in the rectum and release the drug. The composition for rectaladministration may contain an oleaginous base as a suitablenonirritating excipient. Such bases include, without limitation,lanolin, cocoa butter, and polyethylene glycol.

The therapeutic or pharmaceutical composition may include variousmaterials, which modify the physical form of a solid or liquid dosageunit. For example, the composition may include materials that form acoating shell around the active ingredients. The materials that form thecoating shell are typically inert, and may be selected from, forexample, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule. The therapeutic or pharmaceutical compositions in solid orliquid form may include a component that binds to agent and therebyassists in the delivery of the compound. Suitable components that mayact in this capacity include monoclonal or polyclonal antibodies, one ormore proteins or a liposome.

The therapeutic or pharmaceutical composition may consist essentially ofdosage units that can be administered as an aerosol. The term aerosol isused to denote a variety of systems ranging from those of colloidalnature to systems consisting of pressurized packages. Delivery may be bya liquefied or compressed gas or by a suitable pump system thatdispenses the active ingredients. Aerosols may be delivered in singlephase, bi-phasic, or tri-phasic systems in order to deliver the activeingredient(s). Delivery of the aerosol includes the necessary container,activators, valves, subcontainers, and the like, which together may forma kit. One of ordinary skill in the art, without undue experimentationmay determine preferred aerosols.

The compositions described herein may be prepared with carriers thatprotect the agents against rapid elimination from the body, such as timerelease formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, polyorthoesters, polylactic acid and others known to those ofordinary skill in the art.

The therapeutic or pharmaceutical compositions may be prepared bymethodology well known in the pharmaceutical art. For example, atherapeutic or pharmaceutical composition intended to be administered byinjection may comprise one or more of salts, buffers and/or stabilizers,with sterile, distilled water so as to form a solution. A surfactant maybe added to facilitate the formation of a homogeneous solution orsuspension. Surfactants are compounds that non-covalently interact withthe agent so as to facilitate dissolution or homogeneous suspension ofthe agent in the aqueous delivery system.

The therapeutic or pharmaceutical compositions may be administered in atherapeutically effective amount, which will vary depending upon avariety of factors including the activity of the specific compoundemployed; the metabolic stability and length of action of the compound;the age, body weight, general health, sex, and diet of the subject; themode and time of administration; the rate of excretion; the drugcombination; the severity of the particular disorder or condition; andthe subject undergoing therapy. In some instances, a therapeuticallyeffective daily dose is (for a 70 kg mammal) from about 0.001 mg/kg(i.e., ˜0.07 mg) to about 100 mg/kg (i.e., ˜7.0 g); preferably atherapeutically effective dose is (for a 70 kg mammal) from about 0.01mg/kg (i.e., ˜0.7 mg) to about 50 mg/kg (i.e., ˜3.5 g); more preferablya therapeutically effective dose is (for a 70 kg mammal) from about 1mg/kg (i.e., ˜70 mg) to about 25 mg/kg (i.e., ˜1.75 g). In someembodiments, the therapeutically effective dose is administered on aweekly, bi-weekly, or monthly basis. In specific embodiments, thetherapeutically effective dose is administered on a weekly, bi-weekly,or monthly basis, for example, at a dose of about 1-10 or 1-5 mg/kg, orabout 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg.

The combination therapies described herein may include administration ofa single pharmaceutical dosage formulation, which contains an anti-NRP2antibody and an additional therapeutic agent (e.g., immunotherapy agent,chemotherapeutic agent, hormonal therapeutic agent, kinase inhibitor),as well as administration of compositions comprising an anti-NRP2antibody and an additional therapeutic agent in its own separatepharmaceutical dosage formulation. For example, an anti-NRP2 antibody asdescribed herein and additional therapeutic agent can be administered tothe subject together in a single oral dosage composition such as atablet or capsule, or each agent administered in separate oral dosageformulations. Similarly, an anti-NRP2 antibody as described herein andadditional therapeutic agent can be administered to the subject togetherin a single parenteral dosage composition such as in a saline solutionor other physiologically acceptable solution, or each agent administeredin separate parenteral dosage formulations. As another example, forcell-based therapies, an anti-NRP2 antibody can be mixed with the cellsprior to administration, administered as part of a separate composition,or both. Where separate dosage formulations are used, the compositionscan be administered at essentially the same time, i.e., concurrently, orat separately staggered times, i.e., sequentially and in any order;combination therapy is understood to include all these regimens.

Also included are patient care kits, comprising (a) at least oneantibody or antigen-binding fragment thereof that specifically binds toa human neuropilin 2 (NRP2) polypeptide (an anti-NRP2 antibody), asdescribed herein; and optionally (b) at least one additional therapeuticagent (e.g., immunotherapy agent, chemotherapeutic agent, hormonaltherapeutic agent, kinase inhibitor). In certain kits, (a) and (b) arein separate therapeutic compositions. In some kits, (a) and (b) are inthe same therapeutic composition.

The kits herein may also include a one or more additional therapeuticagents or other components suitable or desired for the indication beingtreated, or for the desired diagnostic application. The kits herein canalso include one or more syringes or other components necessary ordesired to facilitate an intended mode of delivery (e.g., stents,implantable depots, etc.).

In some embodiments, a patient care kit contains separate containers,dividers, or compartments for the composition(s) and informationalmaterial(s). For example, the composition(s) can be contained in abottle, vial, or syringe, and the informational material(s) can becontained in association with the container. In some embodiments, theseparate elements of the kit are contained within a single, undividedcontainer. For example, the composition is contained in a bottle, vialor syringe that has attached thereto the informational material in theform of a label. In some embodiments, the kit includes a plurality(e.g., a pack) of individual containers, each containing one or moreunit dosage forms (e.g., a dosage form described herein) of an anti-NRP2antibody and optionally at least one additional therapeutic agent. Forexample, the kit includes a plurality of syringes, ampules, foilpackets, or blister packs, each containing a single unit dose of ananti-NRP2 antibody and optionally at least one additional therapeuticagent. The containers of the kits can be air tight, waterproof (e.g.,impermeable to changes in moisture or evaporation), and/or light-tight.

The patient care kit optionally includes a device suitable foradministration of the composition, e.g., a syringe, inhalant, dropper(e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or anysuch delivery device. In some embodiments, the device is an implantabledevice that dispenses metered doses of the agent(s). Also included aremethods of providing a kit, e.g., by combining the components describedherein.

Bioassays and Analytical Assays for Drug Release Assays and ProductSpecifications, Diagnostics, and Reagents

Also included are bioassays that relate to anti-NRP2 antibodies andrelated agents such as therapeutic and diagnostic reagents. Examplesinclude bioassays and analytical assays that measure purity, biologicalactivity, affinity, solubility, pH, endotoxin levels, among others, manyof which are described herein. Also included are assays that establishdose response curves and/or provide one or more bases for comparisonbetween different batches of antibody. Batch comparisons can be based onany one or more of chemical characterization, biologicalcharacterization, and clinical characterization. Also included aremethods of evaluating the potency, stability, pharmacokinetics, andimmunogenicity of a selected antibody. Among other uses, these and othermethods can be used for lot releasing testing of biologic or chemicalagents, including anti-NRP2 antibodies, described herein.

Certain embodiments include the use of bioaffinity assays. Such assayscan be used to assess the binding affinity, for example, between ananti-NRP2 antibody and at least one NRP2 ligand (for example, an NRP2ligand from Table N2 or Table N3 and/or an HRS polypeptide from TableH1), including its ability to interfere with the interaction between ahuman NRP2 polypeptide and the at least one NRP2 ligand, or othercellular binding partner. Certain exemplary binding affinity assays mayutilize ELISA assays, and other immunoassays as described herein andknown in the art. Certain assays utilize high-performance receptorbinding chromatography (see, e.g., Roswall et al., Biologicals.24:25-39, 1996). Other exemplary binding affinity assays may utilizesurface plasmon resonance (SPR)-based technologies. Examples includeBIACore technologies, certain of which integrate SPR technology with amicrofluidics system to monitor molecular interactions in real time atconcentrations ranging from pM to mM. Also included are KINEXA™ assays,which provide accurate measurements of binding specificity, bindingaffinity, and binding kinetics/rate constants.

Certain embodiments relate to immunoassays for evaluating or optimizingthe immunogenicity of anti-NRP2 antibodies. Examples include ex vivohuman cellular assays and in vitro immuno-enzymatic assays to provideuseful information on the immunogenic potential of a therapeuticprotein. Ex vivo cell-response assays can be used, for example, toreproduce the cellular co-operation between antigen-presenting cells(APCs) and T-cells, and thereby measure T-cells activation after contactwith a protein of interest. Certain in vitro enzymatic assays mayutilize a collection of recombinant HLA-DR molecules that cover asignificant portion of a relevant human population, and may includeautomated immuno-enzymatic assays for testing the binding of peptides(stemming from the fragmentation of the therapeutic protein) with theHLA-DR molecules. Also included are methods of reducing theimmunogenicity of a selected protein, such as by using these and relatedmethods to identify and then remove or alter one or more T-cell epitopesfrom an anti-NRP2 antibody.

Also included are biological release assays (e.g., cell-based assays)for measuring parameters such as specific biological activities,including non-canonical biological activities, and cytotoxicity. Certainspecific biological assays include, for example, cell-based assays thatutilize a cellular binding partner (e.g., cell-surface receptor (forexample a NRP2 polypeptide and/or at least one NRP2 ligand (for example,an NRP2 ligand from Table N2 or Table N3) presented on the cellsurface), which is either endogenously, or recombinantly expressed onthe cell surface), which is functionally coupled to a readout, such as afluorescent or luminescent indicator of NRP2 or NRP2 ligand binding, orfunctional activity, as described herein.

For instance, specific embodiments include a cell that eitherendogenously or recombinantly expresses a human NRP2 polypeptide on thecell surface, which allows assessment of the ability of anti-NRP2antibody to bind NRP2. In some embodiments, the anti-NRP2 antibodyand/or the NRP2 polypeptide is/are functionally coupled to a readout orindicator, such as a fluorescent or luminescent indicator to measure thebinding and/or biological activity of the NRP2 polypeptide. In someembodiments, the cell also expresses at least one NRP2 ligand (forexample, an NRP2 ligand from Table N2 or Table N3 and/or an HRSpolypeptide from Table H1), wherein the at least one NRP2 ligand iscoupled to a readout or indicator, such as a fluorescent or luminescentindicator of binding and/or biological activity of the at least one NRP2ligand.

Also included are in vivo biological assays to characterize thepharmacokinetics of an anti-NRP2 antibody, typically utilizingengineered, or wild type mice, rat, monkey or other mammal (see, e.g.,Lee et al., The Journal of Pharmacology. 281:1431-1439, 1997). Examplesof cytotoxicity-based biological assays include release assays (e.g.,chromium or europium release assays to measure apoptosis; see, e.g., vonZons et al., Clin Diagn Lab Immunol. 4:202-207, 1997), among others,which can assess the cytotoxicity anti-NRP2 antibodies, whether forestablishing dose response curves, batch testing, or other propertiesrelated to approval by various regulatory agencies, such as the Food andDrug Administration (FDA).

Also included are assays for evaluating the effects of an anti-NRP2antibody on immune cells. Examples include an assay system, comprisingan activated population of T-cells and at least one anti-NRP2 antibody,wherein the at least one anti-NRP2 antibody interferes with binding ofNRP2 to at least one NRP2 ligand (for example, an NRP2 ligand from TableN2 or Table N3 and/or an HRS polypeptide from Table H1).

Certain embodiments include an assay system, comprising a singlemonoclonal anti-NRP2 antibody and at least one human NRP2 polypeptide,wherein the anti-NRP2 antibody binds to the NRP2 polypeptide. In someinstances, the at least one antibody comprises an IgG4 Fc domain.

Also included are testing material(s), comprising a purified NRP2polypeptide, wherein said purified NRP2 polypeptide is bound to a solidsubstrate in a manner that enables antibody binding detection.

Such assays and materials can be used, for example, to develop a doseresponse curve for a selected anti-NRP2 antibody, and/or to compare thedose response curve of different batches of proteins or other agents. Adose-response curve is an X-Y graph that relates the magnitude of astressor to the response of a receptor, such as an NRP2-NRP2 ligand (forexample, an NRP2 ligand from Table N2 or Table N3 and/or an HRSpolypeptide from Table H1) interaction; the response may be aphysiological or biochemical response, such as a non-canonicalbiological activity in a cell in vitro or in a cell or tissue in vivo, atherapeutically effective amount as measured in vivo (e.g., as measuredby EC₅₀), or death, whether measured in vitro or in vivo (e.g., celldeath, organismal death). Death is usually indicated as an LD₅₀, astatistically-derived dose that is lethal to 50% of a modeledpopulation, though it can be indicated by LC₀₁ (lethal dose for 1% ofthe animal test population), LC₁₀₀ (lethal dose for 100% of the animaltest population), or LC_(LO) (lowest dose causing lethality). Almost anydesired effect or endpoint can be characterized in this manner.

The measured dose of a response curve is typically plotted on the X axisand the response is plotted on the Y axis. More typically, the logarithmof the dose is plotted on the X axis, most often generating a sigmoidalcurve with the steepest portion in the middle. The No Observable EffectLevel (NOEL) refers to the lowest experimental dose for which nomeasurable effect is observed, and the threshold dose refers to thefirst point along the graph that indicates a response above zero. As ageneral rule, stronger drugs generate steeper dose response curves. Formany drugs, the desired effects are found at doses slightly greater thanthe threshold dose, often because lower doses are relatively ineffectiveand higher doses lead to undesired side effects. For in vivo generateddose response curves, a curve can be characterized by values such aspg/kg, mg/kg, or g/kg of body-weight, if desired.

For batch comparisons, it can be useful to calculate the coefficient ofvariation (CV) between different dose response curves of differentbatches (e.g., between different batches of anti-NRP2 antibody), in partbecause the CV allows comparison between data sets with different unitsor different means. For instance, in certain exemplary embodiments, twoor three or more different batches of anti-NRP2 antibodies or otheragents have a CV between them of less than about 30%, 20%, 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% for a 4, 5, 6,7, or 8 point dose curve. In certain embodiments, the dose responsecurve is measured in a cell-based assay, and its readout relates to anincrease or a decrease in a selected activity of an anti-NRP2 antibody.In certain embodiments, the dose response curve is measured in a cellrelease assay or animal model (e.g., mouse model), and its readoutrelates to cell death or animal death. Other variations will be apparentto persons skilled in the art.

Expression and Purification Systems

Certain embodiments include methods and related compositions forexpressing and purifying an anti-NRP2 antibody or otherpolypeptide-based agent described herein. Such recombinant anti-NRP2antibodies can be conveniently prepared using standard protocols asdescribed for example in Sambrook, et al., (1989, supra), in particularSections 16 and 17; Ausubel et al., (1994, supra), in particularChapters 10 and 16; and Coligan et al., Current Protocols in ProteinScience (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5and 6. As one general example, anti-NRP2 antibodies may be prepared by aprocedure including one or more of the steps of: (a) preparing aconstruct comprising a polynucleotide sequences that encode an anti-NRP2antibody heavy and light chain and that are operably linked to aregulatory element; (b) introducing the constructs into a host cell; (c)culturing the host cell to express an anti-NRP2 antibody; and (d)isolating an anti-NRP2 antibody from the host cell.

Anti-NRP2 antibody polynucleotides are described elsewhere herein. Inorder to express a desired polypeptide, a nucleotide sequence encodingan anti-NRP2 antibody, or a functional equivalent, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al.,Current Protocols in Molecular Biology (1989).

A variety of expression vector/host systems are known and may beutilized to contain and express polynucleotide sequences. These include,but are not limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems, including mammalian cell and more specifically human cellsystems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are needed, vectors which direct highlevel expression of fusion proteins that are readily purified may beused. Such vectors include, but are not limited to, the multifunctionalE. coli cloning and expression vectors such as BLUESCRIPT (Stratagene),in which the sequence encoding the polypeptide of interest may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem.264:5503 5509 (1989)); and the like. pGEX Vectors (Promega, Madison,Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

Certain embodiments may employ E. coli-based expression systems (see,e.g., Structural Genomics Consortium et al., Nature Methods. 5:135-146,2008). These and related embodiments may rely partially or totally onligation-independent cloning (LIC) to produce a suitable expressionvector. In specific embodiments, protein expression may be controlled bya T7 RNA polymerase (e.g., pET vector series). These and relatedembodiments may utilize the expression host strain BL21(DE3), a λDE3lysogen of BL21 that supports T7-mediated expression and is deficient inIon and ompT proteases for improved target protein stability. Alsoincluded are expression host strains carrying plasmids encoding tRNAsrarely used in E. coli, such as ROSETTA™ (DE3) and Rosetta 2 (DE3)strains. Cell lysis and sample handling may also be improved usingreagents sold under the trademarks BENZONASE® nuclease and BUGBUSTER®Protein Extraction Reagent. For cell culture, auto-inducing media canimprove the efficiency of many expression systems, includinghigh-throughput expression systems. Media of this type (e.g., OVERNIGHTEXPRESS™ Autoinduction System) gradually elicit protein expressionthrough metabolic shift without the addition of artificial inducingagents such as IPTG. Particular embodiments employ hexahistidine tags(such as those sold under the trademark HIS•TAG® fusions), followed byimmobilized metal affinity chromatography (IMAC) purification, orrelated techniques. In certain aspects, however, clinical grade proteinscan be isolated from E. coli inclusion bodies, without or without theuse of affinity tags (see, e.g., Shimp et al., Protein Expr Purif.50:58-67, 2006). As a further example, certain embodiments may employ acold-shock induced E. coli high-yield production system, becauseover-expression of proteins in Escherichia coli at low temperatureimproves their solubility and stability (see, e.g., Qing et al., NatureBiotechnology. 22:877-882, 2004).

Also included are high-density bacterial fermentation systems. Forexample, high cell density cultivation of Ralstonia eutropha allowsprotein production at cell densities of over 150 g/L, and the expressionof recombinant proteins at titers exceeding 10 g/L.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al., Methods Enzymol. 153:516-544 (1987). Also included arePichia pandoris expression systems (see, e.g., Li et al., NatureBiotechnology. 24, 210-215, 2006; and Hamilton et al., Science,301:1244, 2003). Certain embodiments include yeast systems that areengineered to selectively glycosylate proteins, including yeast thathave humanized N-glycosylation pathways, among others (see, e.g.,Hamilton et al., Science. 313:1441-1443, 2006; Wildt et al., NatureReviews Microbiol. 3:119-28, 2005; and Gerngross et al.,Nature-Biotechnology. 22:1409-1414, 2004; U.S. Pat. Nos. 7,629,163;7,326,681; and 7,029,872). Merely by way of example, recombinant yeastcultures can be grown in Fernbach Flasks or 15 L, 50 L, 100 L, and 200 Lfermentors, among others.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671-1680(1984); Broglie et al., Science 224:838-843 (1984); and Winter et al.,Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, e.g., Hobbs in McGraw Hill,Yearbook of Science and Technology, pp. 191-196 (1992)).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia cells. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusiacells in which the polypeptide of interest may be expressed (Engelhardet al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)). Alsoincluded are baculovirus expression systems, including those thatutilize SF9, SF21, and T. ni cells (see, e.g., Murphy and Piwnica-Worms,Curr Protoc Protein Sci. Chapter 5:Unit5.4, 2001). Insect systems canprovide post-translation modifications that are similar to mammaliansystems.

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad.Sci. U.S.A. 81:3655-3659 (1984)). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Examples of useful mammalian host cell lines include monkey kidney CV1line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidneyline (293 or 293 cells sub-cloned for growth in suspension culture,Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells(BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2). Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNASUSA 77:4216 (1980)); and myeloma cell lines such as NSO and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B. K. C Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268.Certain preferred mammalian cell expression systems include CHO andHEK293-cell based expression systems. Mammalian expression systems canutilize attached cell lines, for example, in T-flasks, roller bottles,or cell factories, or suspension cultures, for example, in 1 L and 5 Lspinners, 5 L, 14 L, 40 L, 100 L and 200 L stir tank bioreactors, or20/50 L and 100/200 L WAVE bioreactors, among others known in the art.

Also included is the cell-free expression of proteins. These and relatedembodiments typically utilize purified RNA polymerase, ribosomes, tRNAand ribonucleotides; these reagents may be produced by extraction fromcells or from a cell-based expression system.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf. et al., ResultsProbl. Cell Differ. 20:125-162 (1994)).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, post-translationalmodifications such as acetylation, carboxylation, glycosylation,phosphorylation, lipidation, and acylation. Post-translationalprocessing which cleaves a “prepro” form of the protein may also be usedto facilitate correct insertion, folding and/or function. Different hostcells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, in addition tobacterial cells, which have or even lack specific cellular machinery andcharacteristic mechanisms for such post-translational activities, may bechosen to ensure the correct modification and processing of the foreignprotein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for about 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type. Transientproduction, such as by transient transfection or infection, can also beemployed. Exemplary mammalian expression systems that are suitable fortransient production include HEK293 and CHO-based systems.

Any number of selection systems may be used to recover transformed ortransduced cell lines. These include, but are not limited to, the herpessimplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977))and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823(1990)) genes which can be employed in tk- or aprt-cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., PNAS USA. 77:3567-70 (1980)); npt, whichconfers resistance to the aminoglycosides, neomycin and G-418(Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); and als or pat,which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci. U.S.A. 85:8047-51 (1988)). The use of visible markers hasgained popularity with such markers as green fluorescent protein (GFP)and other fluorescent proteins (e.g., RFP, YFP), anthocyanins,β-glucuronidase and its substrate GUS, and luciferase and its substrateluciferin, being widely used not only to identify transformants, butalso to quantify the amount of transient or stable protein expressionattributable to a specific vector system (see, e.g., Rhodes et al.,Methods Mol. Biol. 55:121-131 (1995)).

Also included are high-throughput protein production systems, ormicro-production systems. Certain aspects may utilize, for example,hexa-histidine fusion tags for protein expression and purification onmetal chelate-modified slide surfaces or MagneHis Ni-Particles (see,e.g., Kwon et al., BMC Biotechnol. 9:72, 2009; and Lin et al., MethodsMol Biol. 498:129-41, 2009)). Also included are high-throughputcell-free protein expression systems (see, e.g., Sitaraman et al.,Methods Mol Biol. 498:229-44, 2009). These and related embodiments canbe used, for example, to generate microarrays of anti-NRP2 antibodieswhich can then be used for screening libraries to identify antibodiesand antigen-binding domains that interact with the NRP2 polypeptide(s)of interest.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using binding agents or antibodies suchas polyclonal or monoclonal antibodies specific for the product, areknown in the art. Examples include enzyme-linked immunosorbent assay(ELISA), western immunoblots, radioimmunoassays (RIA), and fluorescenceactivated cell sorting (FACS). These and other assays are described,among other places, in Hampton et al., Serological Methods, a LaboratoryManual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. Certain specific embodiments utilizeserum free cell expression systems. Examples include HEK293 cells andCHO cells that can grown on serum free medium (see, e.g., Rosser et al.,Protein Expr. Purif. 40:237-43, 2005; and U.S. Pat. No. 6,210,922).

An antibody, or antigen-binding fragment thereof, produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides maybe designed to contain signal sequences which direct secretion of theencoded polypeptide through a prokaryotic or eukaryotic cell membrane.Other recombinant constructions may be used to join sequences encoding apolypeptide of interest to nucleotide sequence encoding a polypeptidedomain which will facilitate purification and/or detection of solubleproteins. Examples of such domains include cleavable and non-cleavableaffinity purification and epitope tags such as avidin, FLAG tags,poly-histidine tags (e.g., 6×His), cMyc tags, V5-tags, glutathioneS-transferase (GST) tags, and others.

The protein produced by a recombinant cell can be purified andcharacterized according to a variety of techniques known in the art.Exemplary systems for performing protein purification and analyzingprotein purity include fast protein liquid chromatography (FPLC) (e.g.,AKTA and Bio-Rad FPLC systems), high-pressure liquid chromatography(HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistries forpurification include ion exchange chromatography (e.g., 0, S), sizeexclusion chromatography, salt gradients, affinity purification (e.g.,Ni, Co, FLAG, maltose, glutathione, protein A/G), gel filtration,reverse-phase, ceramic HYPERD® ion exchange chromatography, andhydrophobic interaction columns (HIC), among others known in the art.Also included are analytical methods such as SDS-PAGE (e.g., coomassie,silver stain), immunoblot, Bradford, and ELISA, which may be utilizedduring any step of the production or purification process, typically tomeasure the purity of the protein composition.

Also included are methods of concentrating anti-NRP2 antibodies andantigen-binding fragments thereof, and composition comprisingconcentrated soluble proteins. In different aspects such concentratedsolutions of anti-NRP2 antibodies may comprise proteins at aconcentration of about 5 mg/mL; or about 8 mg/mL; or about 10 mg/mL;about 15 mg/mL; or about 20 mg/mL.

In some aspects, such compositions may be substantially monodisperse,meaning that an at least one anti-NRP2 antibody exists primarily (i.e.,at least about 90%, or greater) in one apparent molecular weight formwhen assessed for example, by size exclusion chromatography, dynamiclight scattering, or analytical ultracentrifugation.

In some aspects, such compositions have a purity (on a protein basis) ofat least about 90%, or in some aspects at least about 95% purity, or insome embodiments, at least 98% purity. Purity may be determined via anyroutine analytical method as known in the art.

In some aspects, such compositions have a high molecular weightaggregate content of less than about 10%, compared to the total amountof protein present, or in some embodiments such compositions have a highmolecular weight aggregate content of less than about 5%, or in someaspects such compositions have a high molecular weight aggregate contentof less than about 3%, or in some embodiments a high molecular weightaggregate content of less than about 1%. High molecular weight aggregatecontent may be determined via a variety of analytical techniquesincluding for example, by size exclusion chromatography, dynamic lightscattering, or analytical ultracentrifugation.

Examples of concentration approaches contemplated herein includelyophilization, which is typically employed when the solution containsfew soluble components other than the protein of interest.Lyophilization is often performed after HPLC run, and can remove most orall volatile components from the mixture. Also included areultrafiltration techniques, which typically employ one or more selectivepermeable membranes to concentrate a protein solution. The membraneallows water and small molecules to pass through and retains theprotein; the solution can be forced against the membrane by mechanicalpump, gas pressure, or centrifugation, among other techniques.

In certain embodiments, the reagents, anti-NRP2 antibodies, or relatedagents have a purity of at least about 90%, as measured according toroutine techniques in the art. In certain embodiments, such asdiagnostic compositions or certain therapeutic compositions, ananti-NRP2 antibody composition has a purity of at least about 95%. Inspecific embodiments, such as therapeutic or pharmaceuticalcompositions, an anti-NRP2 antibody composition has a purity of at leastabout 97% or 98% or 99%. In other embodiments, such as when being usedas reference or research reagents, anti-NRP2 antibodies can be of lesserpurity, and may have a purity of at least about 50%, 60%, 70%, or 80%.Purity can be measured overall or in relation to selected components,such as other proteins, e.g., purity on a protein basis.

Purified anti-NRP2 antibodies can also be characterized according totheir biological characteristics. Binding affinity and binding kineticscan be measured according to a variety of techniques known in the art,such as Biacore® and related technologies that utilize surface plasmonresonance (SPR), an optical phenomenon that enables detection ofunlabeled interactants in real time. SPR-based biosensors can be used indetermination of active concentration, screening and characterization interms of both affinity and kinetics. The presence or levels of one ormore canonical or non-canonical biological activities can be measuredaccording to cell-based assays, including those that utilize a cellularbinding partner of a selected anti-NRP2 antibody, which is functionallycoupled to a readout or indicator, such as a fluorescent or luminescentindicator of biological activity, as described herein.

In certain embodiments, as noted above, an anti-NRP2 antibodycomposition is substantially endotoxin free, including, for example,about 95% endotoxin free, preferably about 99% endotoxin free, and morepreferably about 99.99% endotoxin free. The presence of endotoxins canbe detected according to routine techniques in the art, as describedherein. In specific embodiments, an anti-NRP2 antibody composition ismade from a eukaryotic cell such as a mammalian or human cell insubstantially serum free media. In certain embodiments, as noted herein,an anti-NRP2 antibody composition has an endotoxin content of less thanabout 10 EU/mg of anti-NRP2 antibody, or less than about 5 EU/mg ofanti-NRP2 antibody, less than about 3 EU/mg of anti-NRP2 antibody, orless than about 1 EU/mg of anti-NRP2 antibody.

In certain embodiments, an anti-NRP2 antibody composition comprises lessthan about 10% wt/wt high molecular weight aggregates, or less thanabout 5% wt/wt high molecular weight aggregates, or less than about 2%wt/wt high molecular weight aggregates, or less than about or less thanabout 1% wt/wt high molecular weight aggregates.

Also included are protein-based analytical assays and methods, which canbe used to assess, for example, protein purity, size, solubility, anddegree of aggregation, among other characteristics. Protein purity canbe assessed a number of ways. For instance, purity can be assessed basedon primary structure, higher order structure, size, charge,hydrophobicity, and glycosylation. Examples of methods for assessingprimary structure include N- and C-terminal sequencing andpeptide-mapping (see, e.g., Allen et al., Biologicals. 24:255-275,1996)). Examples of methods for assessing higher order structure includecircular dichroism (see, e.g., Kelly et al., Biochim Biophys Acta.1751:119-139, 2005), fluorescent spectroscopy (see, e.g., Meagher etal., J. Biol. Chem. 273:23283-89, 1998), FT-IR, amide hydrogen-deuteriumexchange kinetics, differential scanning calorimetry, NMR spectroscopy,immunoreactivity with conformationally sensitive antibodies. Higherorder structure can also be assessed as a function of a variety ofparameters such as pH, temperature, or added salts. Examples of methodsfor assessing protein characteristics such as size include analyticalultracentrifugation and size exclusion HPLC (SEC-HPLC), and exemplarymethods for measuring charge include ion-exchange chromatography andisolectric focusing. Hydrophobicity can be assessed, for example, byreverse-phase HPLC and hydrophobic interaction chromatography HPLC.Glycosylation can affect pharmacokinetics (e.g., clearance),conformation or stability, receptor binding, and protein function, andcan be assessed, for example, by mass spectrometry and nuclear magneticresonance (NMR) spectroscopy.

As noted above, certain embodiments include the use of SEC-HPLC toassess protein characteristics such as purity, size (e.g., sizehomogeneity) or degree of aggregation, and/or to purify proteins, amongother uses. SEC, also including gel-filtration chromatography (GFC) andgel-permeation chromatography (GPC), refers to a chromatographic methodin which molecules in solution are separated in a porous material basedon their size, or more specifically their hydrodynamic volume, diffusioncoefficient, and/or surface properties. The process is generally used toseparate biological molecules, and to determine molecular weights andmolecular weight distributions of polymers. Typically, a biological orprotein sample (such as a protein extract produced according to theprotein expression methods provided herein and known in the art) isloaded into a selected size-exclusion column with a defined stationaryphase (the porous material), preferably a phase that does not interactwith the proteins in the sample. In certain aspects, the stationaryphase is composed of inert particles packed into a densethree-dimensional matrix within a glass or steel column. The mobilephase can be pure water, an aqueous buffer, an organic solvent, or amixture thereof. The stationary-phase particles typically have smallpores and/or channels which only allow molecules below a certain size toenter. Large particles are therefore excluded from these pores andchannels, and their limited interaction with the stationary phase leadsthem to elute as a “totally-excluded” peak at the beginning of theexperiment. Smaller molecules, which can fit into the pores, are removedfrom the flowing mobile phase, and the time they spend immobilized inthe stationary-phase pores depends, in part, on how far into the poresthey penetrate. Their removal from the mobile phase flow causes them totake longer to elute from the column and results in a separation betweenthe particles based on differences in their size. A given size exclusioncolumn has a range of molecular weights that can be separated. Overall,molecules larger than the upper limit will not be trapped by thestationary phase, molecules smaller than the lower limit will completelyenter the solid phase and elute as a single band, and molecules withinthe range will elute at different rates, defined by their propertiessuch as hydrodynamic volume. For examples of these methods in practicewith pharmaceutical proteins, see Bruner et al., Journal ofPharmaceutical and Biomedical Analysis. 15: 1929-1935, 1997.

Protein purity for clinical applications is also discussed, for example,by Anicetti et al. (Trends in Biotechnology. 7:342-349, 1989). Morerecent techniques for analyzing protein purity include, withoutlimitation, the LabChip GXII, an automated platform for rapid analysisof proteins and nucleic acids, which provides high throughput analysisof titer, sizing, and purity analysis of proteins. In certainnon-limiting embodiments, clinical grade proteins such as proteinfragments and antibodies can be obtained by utilizing a combination ofchromatographic materials in at least two orthogonal steps, among othermethods (see, e.g., Therapeutic Proteins: Methods and Protocols. Vol.308, Eds., Smales and James, Humana Press Inc., 2005). Typically,protein agents (e.g., anti-NRP2 antibodies, and antigen-bindingfragments) are substantially endotoxin-free, as measured according totechniques known in the art and described herein.

Protein solubility assays are also included. Such assays can beutilized, for example, to determine optimal growth and purificationconditions for recombinant production, to optimize the choice ofbuffer(s), and to optimize the choice of anti-NRP2 antibodies orvariants thereof. Solubility or aggregation can be evaluated accordingto a variety of parameters, including temperature, pH, salts, and thepresence or absence of other additives. Examples of solubility screeningassays include, without limitation, microplate-based methods ofmeasuring protein solubility using turbidity or other measure as an endpoint, high-throughput assays for analysis of the solubility of purifiedrecombinant proteins (see, e.g., Stenvall et al., Biochim Biophys Acta.1752:6-10, 2005), assays that use structural complementation of agenetic marker protein to monitor and measure protein folding andsolubility in vivo (see, e.g., Wigley et al., Nature Biotechnology.19:131-136, 2001), and electrochemical screening of recombinant proteinsolubility in Escherichia coli using scanning electrochemical microscopy(SECM) (see, e.g., Nagamine et al., Biotechnology and Bioengineering.96:1008-1013, 2006), among others. Anti-NRP2 antibodies with increasedsolubility (or reduced aggregation) can be identified or selected foraccording to routine techniques in the art, including simple in vivoassays for protein solubility (see, e.g., Maxwell et al., Protein Sci.8:1908-11, 1999).

Protein solubility and aggregation can also be measured by dynamic lightscattering techniques. Aggregation is a general term that encompassesseveral types of interactions or characteristics, includingsoluble/insoluble, covalent/noncovalent, reversible/irreversible, andnative/denatured interactions and characteristics. For proteintherapeutics, the presence of aggregates is typically consideredundesirable because of the concern that aggregates may cause animmunogenic reaction (e.g., small aggregates), or may cause adverseevents on administration (e.g., particulates). Dynamic light scatteringrefers to a technique that can be used to determine the sizedistribution profile of small particles in suspension or polymers suchas proteins in solution. This technique, also referred to as photoncorrelation spectroscopy (PCS) or quasi-elastic light scattering (QELS),uses scattered light to measure the rate of diffusion of the proteinparticles. Fluctuations of the scattering intensity can be observed dueto the Brownian motion of the molecules and particles in solution. Thismotion data can be conventionally processed to derive a sizedistribution for the sample, wherein the size is given by the Stokesradius or hydrodynamic radius of the protein particle. The hydrodynamicsize depends on both mass and shape (conformation). Dynamic scatteringcan detect the presence of very small amounts of aggregated protein(<0.01% by weight), even in samples that contain a large range ofmasses. It can also be used to compare the stability of differentformulations, including, for example, applications that rely onreal-time monitoring of changes at elevated temperatures. Accordingly,certain embodiments include the use of dynamic light scattering toanalyze the solubility and/or presence of aggregates in a sample thatcontains an anti-NRP2 antibody of the present disclosure.

Although the foregoing embodiments have been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this disclosure that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims. The following examples areprovided by way of illustration only and not by way of limitation. Thoseof skill in the art will readily recognize a variety of noncriticalparameters that could be changed or modified to yield essentiallysimilar results.

EXAMPLES

Reference Sequences NCBI Reference Region and Protein Species Sequencemutations NRP2v1 Human NP_957718.1 NRP2v2 Human NP_003863.2 NRP2v3 HumanNP_958436.1 NRP2v4 Human NP_061004.3 NRP2v5 Human NP_957719.1 NRP2v2crab-eating XP_005574085.1 macaque NRP2v2 Mouse NP_001070872.1 SEMA3FHuman NP_004177.3 19-779 R583A R586A VEGF-C Human NP_005420.1 112-227SEMA3F Mouse NP_001298080.1 19-779 R583A R586A VEGF-C Mouse NP_033532108-223 FLT4 Human NP_891555 PLXNA1 Human NP_115618.3 L839P KDR HumanNP_002244.1 VEGF-A Human NP_001165097.1 27-191

Example 1 Characterization of Humanized, Affinity MaturedAnti-Neuropilin 2 Antibodies

Recombinant, humanized anti-NRP2 antibodies were generated from a panelof murine monoclonal antibodies targeting different domains of humanNRP2. Humanized antibodies were affinity matured to produce highlyselective, potent antibodies with sub nanomolar affinities in thecontext of primarily human IgG constant domain to limit the potentialfor immunogenicity in humans. Initial assessments of binding affinity asmeasured by surface plasmon resonance (SPR), and flow cytometry (FACS)binding to cell lines expressing recombinant NRP2, is presented in TableE1.

TABLE E1 Antibodies Target Affinity EC50 Antibody Isotype domain (nM)(nM) aNRP2-1v3 hIgG4 b2 17 n.d. aNRP2-2v4 hIgG4 c 2.7 n.d. aNRP2-10v5hIgG4 b1 0.20 n.d. aNRP2-10v10 hIgG4 b1 0.029 0.09 aNRP2-10v13 hIgG4-YTEb1 0.358 0.65 aNRP2-11v7 hIgG4 b2 0.95 6.3 aNRP2-14v9 hIgG4 a2 0.15 1.2aNRP2-14v10 hIgG4 a2 0.17 47.5 aNRP2-28v2/4 mIgG1(D265A) b1 0.0599 1.58

Humanization and affinity maturation. Humanized murine monoclonalantibodies were prepared via grafting of murine CDRs onto human IgG4with the selective replacement of murine specific amino acidsubstitutions compared to the corresponding human sequences whennecessary to stabilize the antibody, and screening to confirm binding toNRP2 polypeptides. Humanized antibodies were affinity matured via thesystematic mutation of their CDR sequences, and the most advantageousrecombination of the higher affinity combinations identified were fullycharacterized. Recombinant antibodies were purified from conditionedmedium starting at 2 weeks of culture by flowing over a Protein Aaffinity column, eluting and storing in Phosphate Buffered Saline(1×PBS), pH 7.4. Each lot was tested for protein concentration, purity,and endotoxin level. Purity by SDS-PAGE was routinely >90%.

Anti-NRP2 antibody binding and affinity measurements. Surface plasmonresonance (SPR) methods were used to demonstrate binding of anti-NRP2antibodies to human NRP2 antigen and to measure binding affinities assummarized in Table E1. SPR experiments were conducted on a Bio-RadProteOn XPR36 Protein Interaction Array instrument. Biotinylatedanti-human antibody (ThermoFisher Cat #7103302100) was immobilized onProteOn NLC sensor chips. Anti-NRP2 antibodies were subsequently flowedover and captured by the anti-human antibody. Human NRP2 antigen proteinwas flowed over the captured antibody at varying concentrations (150,50, 16.67, 5.56, 1.85 nM). The sensor chip surface was regeneratedbetween each analyte run to remove anti-NRP2 antibodies and NRP2protein. Data was double referenced against a surface with no anti-NRP2antibody captured (immobilized anti-human antibody only) and a bufferonly blank. Affinity constants were derived by globally fittingsensograms to a Langmuir (1:1) interaction model in the ProteOn managersoftware. For each anti-NRP2 antibody, data from the multiple NRP2concentrations was fit as a single data set with dissociation rateconstant (k_(d)), association rate constant (k_(a)) and R_(max) valuesas global parameters. The reported binding affinity is the equilibriumdissociation constant (K_(D)) calculated from k_(d)/k_(a).

-   -   Running buffer: 50 mM HEPES, 300 mM NaCl, 5 mM CaCl₂, 0.005%        Tween-20, pH 7.4    -   Immobilized antibody: CaptureSelect Human Fab-kappa Kinetics        Biotin Conjugate, ThermoFisher Cat #7103302100    -   Antigen: Human NRP2 (aa23-855) with C-terminal Avi-, Myc- and        His-tags    -   Regeneration buffer: 10 mM glycine pH 2.0

Purification of plasmid DNA. Plasmid containing human NRP2 werepurchased from Origene. To generate a large stock of plasmids and purifythe DNA, plasmid was transformed into chemically competent E. coli cells(One Shot TOP10) according to the manufacturer's instructions. Thetransformed bacteria were grown in the recommended liquid medium (DifcoDehydrated Miler Luria-Bertani medium powder resuspended in water)containing kanamycin monosulfate (50 μg/mL) as an antibiotic. Theplasmid DNA was then purified using the QIAGEN DNA maxi prep kitaccording to the kit's instructions. DNA concentration and purity weremeasured on a spectrophotometer (Nanodrop 2000). An A260/A280 absorbanceratio between 1.8 and 2.0 was required for transfection.

Generation of Expi293-hNRP2 clonal cells stably over-expressing humanNRP2. A plasmid (Origene Technologies Cat #RC220706) encoding the humanNRP2 variant 2 transcript NM_003872 (hNRP2) fused to a Myc-DDK tag waspurchased. The vector was PCR amplified using Q5 polymerase (New EnglandBiolabs Cat #M0491) with the following primer pairs:

(SEQ ID NO: 149) 5′-TGAGGATGACAAAGATTTGCAGCT-3′ (SEQ ID NO: 150)5′-ACCGCGGCCGGCCGTTTATGCCTCGGAGCAGCACTT-3′ (SEQ ID NO: 151)5′-AGTGCCAAGCAAGCAACTCAAA-3′ (SEQ ID NO: 152)5′-AAGTGCTGCTCCGAGGCATAAACGGCCGGCCGCGGT-3′

The resulting PCR products were then fused, cut with MfeI/AgeI (NewEngland Biolabs Cat #R3589, R3552), and ligated into a vector fragmentof RC220706 cut with the same enzymes. This vector, containing anuntagged human NRP2 transcript, was then linearized and re-suspended in10 mM Tris-0.1 mM EDTA. Suspension Expi293 cells, (a HEK293 basedtransient expression system which is maintained in suspension culturesfrom ThermoFisher; Cat #A14527), were grown in expression medium(ThermoFisher Cat #A1435101) at 37° C. and 8% CO₂. The linearizedplasmid described above was transfected into Expi293 cells using an SFCell Line 4D-Nucleofector® X Kit L (Lonza Cat #V4XC-2012) and standardprotocol T-030 for suspension HEK293 cells. Cells were allowed torecover in static culture for 17 hours, transferred to suspension andrecovered an additional 72 hours, and then were selected with 200-350μg/mL G418 in 50 μg increments (ThermoFisher Cat #10131035). Celldensities and viabilities were monitored for a period of 3 weeks, withfresh media/antibiotic replacement every 2-3 days.

To select human NRP2 over-expressing clonal cells, cells were suspendedinto 96-well plates by limited dilution. Single colonies weretransferred to falcon tubes for further expansion under maintainedselection pressure. Clonal cells that over-express human NRP2 werevalidated by a-NRP2 staining followed with flow cytometry analysis.

Binding of anti-NRP2 antibodies to Expi293 cells expressing human NRP2(hNRP2) clonal cells. Expi293-hNRP2 clonal cells were collected bycentrifugation at 300 g for 5 minutes, and washed twice by DPBS (withcalcium and magnesium, ThermoFisher Cat #14040133). Washed cells wereadded to a 96-well V-bottom plate (ThermoFisher Cat #1424572) at 100,000cells/well in 50 μL of DPBS added with Zombie Violet viability stain(Biolegend Cat #423114, diluted at 1:500). From this step on, cells wereprotected from light. Cells were kept at room temperature for 10 min andpelleted down at 300 g for 5 minutes at 4° C. Supernatants containingZombie Violet were discarded, and without washing, 30 uL of antibodiesdiluted in the flow wash buffer (FWB, DPBS plus 2% FBS and 0.1% sodiumazide to inhibit receptor internalization) were added to the cells. Foranti-NRP2 antibody staining on Expi293-hNRP2 clonal cells, in-househuman anti-NRP2 antibodies were tested at final concentrations of0.006-100 nM at 4-fold dilutions. An isotype control human IgG4 antibody(Biolegend Cat #403701) was used instead of the anti-NRP2 antibody todemonstrate the specificity of the staining. Binding was allowed toproceed on ice for 40-60 minutes. Cells were then pelleted at 300 g for5 minutes at 4° C., supernatants removed, and cells washed twice byadding 150 μL of FWB and centrifuging the cells again in the sameconditions. To detect a-NRP2 binding on the cell surface, Cy3-conjugatedgoat anti-human IgG Fc (Jackson ImmunoResearch Cat #109165098) was addedto the cells at a final concentration of 3 μg/mL in 30 μL of FWB. After30-40 minutes of incubation on ice, cells were pelleted at 300 g for 5minutes at 4° C. and supernatants removed, and the cells were washedtwice as described previously. Cells were then resuspended in FWB beforeacquisition on the Cytoflex. Gains were set based on staining controls.Cells were collected with typically more than 10,000 events. Cells werethen analyzed on FlowJo analysis software by excluding dead cells andgating singlets. The statistical analysis was performed using GraphPadPrism. A four-parameter variable-slope curve was fitted to the data([agonist] vs. response) using non-linear regression, and the EC₅₀ andr2 for each curve was determined.

The EC₅₀ of four anti-NRP2 antibodies directed against different domainsof NRP2, were measured for binding to Expi293-hNRP2 clonal cells (seeTable E1 and FIG. 3 ). They showed specific binding to NRP2 withEC_(50s) in the subnanomolar or nanomolar range. By contrast, no bindingwas observed by the isotype control human IgG4 antibody.

Example 2 Binding of Anti-Human Neuropilin 2 Antibodies to CynomolgusMonkey Nrp2

Cross reactivity of the anti-NRP2 antibodies to cynomolgus monkey NRP2was assessed by surface plasmon resonance (SPR) on recombinant proteins,and flow cytometry (FACS) binding to NRP2 expressed on the surface ofHEK 293 cells. Establishing cross reactivity between cynomolgus monkeyand human NRP2 is an important therapeutic development consideration toensure that potential therapeutic candidates can be readily assessed fortoxicity in animal studies; particularly if there is little or no crossreactivity of the antibodies between human and rodent NRP2.

Plasmid DNA. Plasmids containing human or cynomolgus monkey NRP2 werepurchased from Origene. Plasmid propagation and purification wasperformed as described for the human NRP2 plasmid.

Expi293 propagation and transient transfection. Expi293 cells, a HEK293transient expression system maintained in suspension cultures, were usedto express cynomolgus monkey NRP2. Cells were grown in 60 mL Expi293medium within 250 mL vented suspension culture flasks. The propagationof cells was carried out in a Multitron Cell incubator at 37° C., with8% CO₂, 80% humidity, and shaking at 225 rpm. During the maintenance andexpansion phase, Expi293 cells were split twice a week at 0.3×10⁶cells/mL in order to keep the density within the optimal range fortransfection and the viability high. Cell densities and viabilities weredetermined using a cell counter (Cedex HiRes Cell Analyzer).

The day prior to DNA transfection, the Expi293 cells were seeded at adensity of 2.0×10⁶ cells/mL in order to maintain high viability (>95%)and low density (not to exceed 3-5×10⁶ cells/mL), thereby providingcells with fresh nutrients and avoiding transfection impediments causedby any secreted substances.

Prior to DNA transfection, Expi293 cells were counted and reseeded at2.5×10⁶ cells/mL in 50 mL shake flasks (TPP TubeSpin bioreactor tubes).The transfection procedure was performed using the Expifectamine kit.The manufacturer's protocol was adapted for a transfection in a totalvolume of 5 mL instead of 30 mL (i.e., all volumes were divided by afactor of 6). Five pg of each plasmid DNA was diluted in Opti-MEMreduced serum medium, complexed with Expifectamine transfection reagent,and transfected into Expi293 cells. The transfected pools were culturedwith shaking at 225 rpm to ensure complete suspension of the cellswithin the 50 mL bioreactors. Following the manufacturer's protocol,Expifectamine kit enhancers were added after 16-18 hours, and thecultures analyzed by flow cytometry 2 days post-transfection.

Binding of a-NRP2 to cynomolgus monkey NRP2-transfected Expi293 cells.Cynomolgus monkey (cyno) NRP2-transfected Expi293 cells were collectedby centrifugation at 300 g for 5 minutes, and washed twice by DPBS (withcalcium and magnesium, ThermoFisher Cat #14040133). Washed cells wereadded to a 96-well V-bottom plate (ThermoFisher Cat #1424572) at 100,000cells/well in 50 μL of DPBS added with Zombie Violet viability stain(Biolegend Cat #423114, diluted at 1:500). From this step on, cells wereprotected from light. Cells were kept at room temperature for 10 min andpelleted down at 300 g for 5 minutes at 4° C. Supernatants containingZombie Violet were discarded, and without washing, 30 uL of antibodiesdiluted in the flow wash buffer (FWB, DPBS plus 2% FBS and 0.1% sodiumazide to inhibit receptor internalization) were added to the cells. Fora-NRP2 staining on Cyno NRP2-transfected Expi293 cells, in-house humananti-NRP2 antibodies were tested at final concentrations of 0.006-100 nMat 4-fold dilutions. For each test, NRP2 over-expression on the cellsurface was validated by a-NRP2 staining (10 μg/mL R&D #AF567 a-NRP2) inseparate wells. Binding was allowed to proceed on ice for 40-60 minutes.Cells were then pelleted at 300 g for 5 minutes at 4° C., supernatantsremoved, and cells washed twice by adding 150 μL of FWB and centrifugingthe cells again in the same conditions. To detect a-NRP2 binding on thecell surface, Cy3-conjugated goat anti-human IgG Fc or AF647-conjugateddonkey anti-goat IgG (Jackson ImmunoResearch Cat #109165098 & 705605147)was added to the cells at a final concentration of 3 μg/mL in 30 μL ofFWB. After 30-40 minutes of incubation on ice, cells were pelleted at300 g for 5 minutes at 4° C. and supernatants removed, and the cellswere washed twice as described previously. Cells were then resuspendedin FWB before acquisition on the Cytoflex. Gains were set based onstaining controls. Cells were collected with typically more than 10,000events. Cells were then analyzed on FlowJo analysis software byexcluding dead cells and gating singlets.

As additional controls, the following conditions were tested: 1) Anisotype control human IgG4 antibody (BioLegend Cat #403701) was usedinstead of the anti-NRP2 antibody to demonstrate the specificity of thestaining; and 2) Staining with R&D #AF567 a-NRP2 was also determinedusing mock-transfected Expi293 cells to demonstrate over-expression ofCyno NRP2 on Expi293 cells. The statistical analysis was performed usingGraphPad Prism. A four-parameter variable-slope curve was fitted to thedata ([agonist] vs. response) using non-linear regression, and the EC₅₀and r2 for each curve was determined.

The EC₅₀ of three anti-NRP2 antibodies, each against a distinct domainof NRP2, were measured for binding to Cyno NRP2-transfected Expi293cells (see Table E2 and FIG. 4 ). They all showed specific binding toNRP2 with EC_(50s) in the nanomolar range. By contrast, no binding wasobserved by the isotype control human IgG4 antibody.

Anti-NRP2 antibody binding and affinity measurements. Surface plasmonresonance (SPR) methods were used to demonstrate binding of anti-NRP2antibodies to cynomolgus NRP2 antigen and to measure binding affinitiesas summarized in Table E2. SPR experiments were conducted on a Bio-RadProteOn XPR36 Protein Interaction Array instrument. Biotinylatedanti-human antibody (ThermoFisher Cat #7103302100) was immobilized onProteOn NLC sensor chips. Anti-NRP2 antibodies were subsequently flowedover and captured by the anti-human antibody. Cynomolgus NRP2 antigenprotein was flowed over the captured antibody at varying concentrations(150, 50, 16.67, 5.56, 1.85 nM). The sensor chip surface was regeneratedbetween each analyte run to remove anti-NRP2 antibodies and NRP2protein. Data was double referenced against a surface with no anti-NRP2antibody captured (immobilized anti-human antibody only) and a bufferonly blank. Affinity constants were derived by globally fittingsensograms to a bivalent analyte interaction model in the ProteOnmanager software. For each anti-NRP2 antibody, data from the multipleNRP2 concentrations was fit as a single data set with dissociation rateconstant (k_(d)), association rate constant (k_(a)) and R_(max) valuesas global parameters. The reported binding affinity is the equilibriumdissociation constant (K_(D)) calculated from k_(d)/k_(a).

-   -   Running buffer: 50 mM HEPES, 300 mM NaCl, 5 mM CaCl₂, 0.005%        Tween-20, pH 7.4    -   Immobilized antibody: CaptureSelect Human Fab-kappa Kinetics        Biotin Conjugate, Thermo Fisher Cat #7103302100    -   Antigen: Cynomolgus NRP2 (aa23-854) with C-terminal Fc-tag    -   Regeneration buffer: 10 mM glycine pH 2.0

TABLE E2 Comparison of binding affinity to cynomolgus monkey NRP2 DomainEC50 Affinity Antibody Specificity (nM) (nM) aNRP2-14v9 a2 2.8 0.017aNRP2-10v10 b1 2.9 0.032 aNRP2-11v7 b2 5.7 12 Isotype control antibodyn.d. n.d. n.d.

The results from these studies demonstrate that each of the testedantibodies showed comparable binding affinities to human and cyno NRP2,which affinities that were all low nanomolar and within about 5-6 foldof each other between species.

Example 3 Characterization of Anti-Nrp2 Antibodies on Receptor Nrp2Dimerization

To further extend the assessment of the biological activity of theanti-NRP2 antibodies, their activity was assessed in a receptordimerization assay. In brief, vectors encoding a split luciferasepBiT1.1 and pBiT2.1 were obtained from Promega corporation (Madison,Wis.). The complete extracellular domain and transmembrane helices ofNRP2a (version 2), NRP2b (version 5) (See table N1), FLT4 (VEGFR3), KDR(VEGFR2), and plexin A1 (PLXNA1) were cloned into the vectors andscreened for optimal orientation, receptor density, and ligand induceddimerization, following established methods. Expi293 cells (Fisher) weretransfected at 1 million cells/mL 20 hours prior to the assay with NRP2and a co-receptor at equal mass amounts. Cells were counted, and 100,000live cells were plated in a well of a white luminometer plate in LiveCell Imaging Solution (Fisher). Nano-Glo assay substrate (Promega) wasadded, and the plate was read on a Glomax 96 luminometer at roomtemperature to establish baseline luminescence. Antibodies were added tothe well at 100 nM and the plate was again read to establish a newbaseline and monitor the effects of the antibodies on spontaneousreceptor dimerization. Depending on the receptor pair, typically 200 nMSEMA3F-p95 (aTyr Pharma), 2 nM VEGF-C(R&D Systems, Minnesota), or 2 nMVEGF₁₆₅ (R&D Systems, Minnesota) were then added and the plate was againread to measure dimerization of receptors. Normalized responses for eachindividual well were calculated to the time point prior to addition ofligand, then normalized to no-antibody/no-ligand (baseline signal).Replicates were then processed to give average response and standarddeviation. Results are summarized in Table E3 below, and showngraphically in FIGS. 3-10 .

TABLE E3 Receptor Dimerization blocking summary: Domain Ability to BlockAbility to Block Ability to Block Antibody specificity NRP2/PLXNA1NRP2/FLT4(VEGFR3) NRP2/KDR(VEGFR2) aNRP2- a2 Very strong None (Does notNone (Does not 14v9 (80-100% inhibition) substantially block)substantially block) aNRP2- b1 Weak/None Very strong Completely 10v10(Does not substantially (80-100% inhibition) (80-100% inhibition) block)aNRP2- b2 Enhances receptor Partial blocking Enhances receptor 11v7complex formation (20-80% inhibition) complex formation (greater than20%) (greater than 20%) NRP2^(B) b1 Enhances receptor None (Does notNone (Does not complex formation substantially block) substantiallyblock) (greater than 20%)

The results demonstrate that all of the tested antibodies showdifferential functional activity in these assays. Surprisingly specificantibodies show extremely specific and non-obvious functionaldifferentiation at the level of receptor dimerization, demonstratingthat specific epitopes have unique functional roles in this receptorsystem.

For example, antibody aNRP2-14v9, which binds to the a2 domain of NRP2,was demonstrated to exhibit extremely potent inhibition of NRP2-plexinheterodimerization, while having no significant impact on NRP2-VEGFR3heterodimerization. These characteristics suggest that this antibodywould potently inhibit semaphorin 3 signaling through NRP2, withoutsignificantly inhibiting the signaling through VEGFR2 or 3. Suchcharacteristics therefore make this antibody well suited forapplications where the selective modulation of semaphorin signaling isrequired. For example, for modulating effects of semaphorin signaling onaxonal growth avoidance, lymphatic sprouting, cell migration, smoothmuscle contractility, immune cell activation and exhaustion, as moregenerally modulating cell growth and proliferation.

Additionally, antibody aNRP2-10v10, which binds to the b1 domain ofNRP2, shows the ability to potently inhibit NRP2-VEGFR3heterodimerization, without significantly inhibiting NRP2-plexinheterodimerization. These characteristics suggest that this antibodywould potently inhibit VEGF signaling through NRP2, withoutsignificantly inhibiting the signaling through the plexinreceptor/semaphorin 3 pathways. Such characteristics therefore make thisantibody well suited for applications where the selective modulation ofVEGF signaling is required. For example, for modulating effects of VEGFon lymphangiogenesis, neovascularization, angiogenesis, cancer cellgrowth, cancer initiation, cancer migration, cancer cell adhesion,invasion, chemoresistance development and metastasis. Importantly thisantibody does not significantly impair semaphorin 3 signaling, making ituniquely suited for applications where endogenous semaphorin signalingmay be advantageous—for example in any of the applications listed above.

By contrast antibody clone aNRP2-11v7, which binds to the b2 domain ofNRP2, shows the ability to inhibit the heterodimerization of NRP2 toFLT4, but increased the ligand induced dimerization signal for bothPlexin and KDR. Suggesting that it stabilizers heterodimeric and higherorder complexes of NRP2. These characteristics suggest that thisantibody could have unique context dependent signaling effects toinhibit FLT4 signaling while promoting signaling through alternativeNRP2 dependent pathways.

Most surprisingly, in relation to antibody aNRP2-10v10, the previouslydescribed NRP2B antibody (Caunt et al., Cancer Cell. 13:331-342, 2008),shows no ability to inhibit ligand induced dimerization for any of thetested receptor pairs, while enhancing the ligand induced dimerizationof the NRP2/PLXNA1 receptor pair in this assay system. Thesedifferential effects of the NRP2^(B) suggests that it binds to a uniqueepitope of NRP2 compared to aNRP2-10v10, and has functionally distincteffects.

Example 4 Epitope Mapping of aNRP2-10v10 Antibody

Human NRP2a (version 2; see Table N1) and mouse NRP2v2 cDNAs wereobtained from Origene. Because we have previously demonstrated that theaNRP2-10v10 antibody binds specifically to the b1 domain of human NRP2,but not mouse NRP2, mutations in the b1 domain of mouse NRP2 sequence(F299Y, K354N, I383V, M400A, I407V, and L416S) were made in isolation torestore the amino acids which differ between the human and mouseproteins in this region. Human and mouse NRP2, expression vectors, alongwith expression vectors encoding all 6 mutants were transfected intoExpi293 cells at 1 million cells/mL, by standard techniques.Approximately 40 hours post transfection cells were co-stained withaNRP2-10v10, and a species agnostic anti-NRP2 a1 specific bindingantibody. Cells were gated on live, single, NRP2 expressing cells (a1binder positive), as shown in FIG. 11A, and the degree of aNRP2-10v10staining was quantified as mean fluorescent intensity (MFI), as shown inFIG. 11B.

The mutations F299Y, K354N, and L416S restored partial, but incomplete,binding activity of aNRP2-10v10 to mouse NRP2, while mutation of theremaining residues, I383V, M400A, and I407V showed no improvement inbinding (see FIG. 11B). All substitutions that improve binding ofaNRP2-10v10 to mouse NRP2 map on a shared surface of the b1 domain inclose proximity in the published human NRP2 PDB structure 2QQK. Thethree residues map 15.7, 8.9, and 14.3 angstroms apart in the structure,and form a shared epitope. Y299, N354 and S416 also surround the bindingpocket (“C-wall”) for the C-terminus of VEGF-C, and are shownsurrounding T319 which forms the base of the binding pocket (see FIG. 12), where previous work has shown a mutation T319R, blocks binding ofVEGF-C (Parker 2015).

Example 5 Inhibition of Anchorage-Independent Growth and Sensitizationof Tumor Cells to Chemotherapy by aNRP2-10v10

Anchorage-independent growth is the ability of transformed cells to growindependently of a solid surface, and is a hallmark of carcinogenesis.The soft agar colony formation assay is a well-established method forcharacterizing this capability in vitro (J Vis Exp. 2014; (92): 51998).Human triple negative breast cancer (TNBC) cell lines MDA-MB-231(HTB-26™) and BT549 (HTB122™) were obtained from ATCC and employed inthis assay.

MDA-MB-231 was cultured in 1×RPMI media containing 10% FBS and 1% P/S.BT549 was cultured in 1×RPMI media containing 10% FBS, 0.023 U/mLinsulin and 1% P/S. Cells were recovered and expanded from frozen stocksfor 2-3 passages. The NRP2-high population was sorted out on FACSequipment following cell surface staining by a fluorophore-conjugatedanti-NRP2 antibody. The colony formation assay was performed using thesoft agar colony formation fluorometric assay kit obtained from CellBiolab (#CBA-130-CB). Briefly, the sorted NRP2-high cells were suspendedas single cell suspensions, and seeded at 1-1.5 K cells/96-well in the3D soft agar matrix. After overnight incubation to stabilize the cells,the treatment reagents were spiked into the culture media. The chemodrug Cisplatin or 5-FU was added at a final concentration of IC30 or ½IC30, based on a preliminary test that determined their inhibitioncurves on MDA-MB-231 cells, in combination with the antibodiesa-NRP2-10v10 or its isotype control hIgG4 at 100 nM. Reagents wererenewed by spiking-in every 3 days. After 6 or 9 days of treatment, thesoft agar matrix was dissolved and the formed cell colonies werequantified using CyQuant dye diluted in PBS (provided in the Cell Biolabkit), which produced a fluorescence signal proportional to viable cells.

The NRP2-high population of TNBC cells were sorted for use in the colonyformation assay because NRP2 is expressed preferentially on breastcancer stem cells (CSCs) and VEGF/NRP2 signaling was shown to beimportant for the genesis of TNBCs and tumor initiation (EMBO Mol Med.2013 5(4):488-508). Compared to the hIgG4 control, the a-NRP2-10v10antibody in combination with either Cisplatin or 5-FU showed asignificant inhibition of colony formation by MDA-MB-231 cells (FIG.13A). Similar effect was observed for the BT549 cells at certain dosesof Cisplatin or 5-FU. (FIG. 13B). In a separate test with aNRP2-10v10treatment alone, no obvious effect in the colony formation assay wasobserved with these cells. The results suggest that aNRP2-10v10sensitized the TNBC CSC-like cells to chemo drug treatment and has thepotential to be developed in a combination therapy in combating cancer.

Example 6 Inhibition of Anchorage Independent Growth and Sensitizationto Cisplatin and Bevacizumab Therapy by aNRP2-10V10

The immunostaining of TNBC MDA-MB-231 cells revealed that >90% of thesecells exhibit a cancer stem cell (CSC)-like phenotype (CD44+/CD24−),therefore we further performed the colony formation assay using parentalMDA-MB-231 cells without pre-sorting. The matrix for 3D colony formationassay was changed to methylcellulose which is more solid, better mimicsthe extracellular matrix in vivo, and shows better signal-to-backgroundthan soft agar. Other experimental details are essentially the same asdescribed in the previous example.

The results demonstrated that the anti-NRP antibody aNRP2-10v10 incombination with either the chemo drug Cisplatin or the anti-VEGF-A drugBevacizumab, showed a significantly enhanced inhibition of colonyformation by MDA-MB-231 cells, compared to either treatment with theisotype control hIgG4 antibody (see FIG. 14 ). aNRP2-10v10 thussignificantly augments the effectiveness of both cisplatin andBevacizumab in treating triple negative breast cancer.

Example 7 Inhibition of Cell Migration of Human Lymphatic EndothelialCell (HLEC) Migration Toward Vascular Endothelial Growth Factor C(VEGF-C) by Antibodies aNRP2-10V10 and aNRP2-11V7

Migration assays were performed using a modified Boyden Chamber with 8μM pore size membrane, 16-well CIM plate (ACEA Biosciences.) Both sidesof the membrane were coated with 5 ug/ml Fibronectin (Sigma) for 30minutes at 37° C., 5% CO₂. 160 μl of 191.5 pM VEGF-C or assay medium(0.1% BSA, ECM) was added to the lower chamber, the upper well attached,50 ul assay medium added to the upper chamber before replacing the lid,and the plate was incubated for 1 hour at 37° C., 5% CO₂ before baselinemeasurement was taken. HLECs were harvested, washed, and re-suspended inassay medium at 100,000 cells/well with aNRP2 antibody or controlproteins and incubated for 30 minutes at 37° C., 5% CO₂. Cells wereadded to the plate and incubated at room temperature for 30 minutes.Plate was inserted into xCELLigence RTCA DP (ACEA Biosciences)instrument, and migration was measured real-time every 5 minutes over a24 hour period (2 wells per condition).

The results demonstrated that the anti-NRP2, VEGF-C blocking antibodies,aNRP2-10v10, and aNRP2-11v7 were able to effective block the migrationof HLEC cells towards VEGF-C in a dose dependent manner (see FIG. 15 ).HLECs were inhibited by both NRP2 antibodies to levels consistent withpositive control antibodies (αKDR or 3C5) directed against VEGFR2 orVEGFR3 respectively.

Example 8 Blocking of Semaphorin 3F Mediated Inhibition of AKTPhosphorylated by Antibodies aNRP2-14V10 and aNRP2-11V7, but notaNRP2-10V10

As shown schematically in FIG. 17 , the treatment of cells with Sema3Fshould specifically block PI3K activity via NRP2 mediated interactionwith plexin A1, thereby resulting in reduced phospho-Akt levels. Todirectly test the ability of the anti-NRP2 antibodies to block thiseffect, lysates were prepared following standard protocols (MesoScaleDiscovery, Phospho(Ser473)/Total Akt Assay Whole Cell Lysate Kit#K15100D). Serum starved and confluent U251 cells (Sigma, Cat. 09063001)were treated with 10 nM rapamycin (Sigma, Cat. 553211, 30 minutes), andblocking antibodies or controls were added (10 ug/mL, 30 minutes),followed by 25 nM Sema3F or serum free media for 1 hour in triplicate.Lysate protein concentrations were determined by BCA assay and sampleswere equalized to 0.4 mg/mL. Phospho and Total AKT were measuredfollowing the manufacturer's instructions and percent phosphorylationwas calculated by dividing phosphorylated AKT relative units by totalAKT relative units. Blocking of Sema3F for each antibody or control wasdetermined by comparing percent phosphorylation of Sema3F treated cellsto percent phosphorylation of non-Sema3F treated cells.

Treatment of U251 cells with Sema3F resulted (see FIG. 16 ) in anapproximately 50% decrease in intracellular phospho-Akt levels that canbe blocked by both the semaphorin blocking antibodies aNRP2-11v7 andaNRP2-14v10, but is not blocked by the VEGF-C blocking antibodyaNRP2-10v10 or with mIgG1 and hIgG4 control antibodies.

Example 9 Evaluation of Anti-Tumor Activity of Test Compound aNRP2-28 onB16-F10 Mouse Melanoma in C57BL/6 Mice

Studies were performed to investigate the in vivo anticancer potentialof antibody aNRP2-10v10 using a surrogate antibody, aNRP2-28, (whichrecognizes mouse NRP2 at a similar epitope region as the antibodyaNRP2-10v10 (which does not cross react with mouse NRP2) in a syngeneicmouse model using B16-F10 cells (a mouse melanoma cancer model) preparedas described below.

Animals and Animal husbandry. All mice were ear tagged foridentification purpose. Upon arrival, animals were examined to ensurethat they were healthy. The animals were housed in autoclaved solidfloor polycarbonate cages. Housing and sanitation were performedaccording to industry standards. All animal handling was performed in alaminar flow hood located in a clean room. In all experiments,euthanasia criteria were set for an upper limit on subcutaneous tumorvolume (usually >2,000 mm³) and body weight loss (usually >20% bodyweight loss for an individual during the study). Animal work wasperformed at either Comparative Biosciences, Inc., 786 Lucerne Dr.,Sunnyvale, Calif. 94085 or at aTyr Pharma, 3545 John Hopkins Court,Suite 250, San Diego, Calif. 92121. The test antibody dosing regimen wasinitiated once the mean tumor volume reached 50-100 mm³. Tumormeasurements were recorded two times a week until study termination, asdescribed below.

Tumor monitoring. Once palpable, sub-cutaneous tumors were measuredthree times a week with a digital caliper. Tumor volumes were calculatedusing formula:Tumor Volume=length×width×width×½Statistical analysis. Data are expressed as mean±SEM o. In experimentsin which animals were euthanized due to tumor burden or body weightloss, the terminal tumor volume was carried forward for statisticalanalysis. In cases where an animal was found dead but did not have alarge tumor (cause of mortality unknown), the animal's data were removedprior to statistical analysis. Significance of difference over time wastested with a 2-way repeated measures ANOVA followed by Dunnett'spost-hoc test. Group comparisons were conducted using 1-way ANOVA(parametric or Kruskal Wallis, as noted in the figure legends). A pvalue<0.05 was considered significant.

Cell culture and implantation. Mouse B16-F10 cell line was purchasedfrom ATCC. The cells were cultured in 75 cm² flasks containing DMEMmedia supplemented with 10% fetal bovine serum (FBS) and incubated at37° C. in humidified atmosphere of 5% CO₂. As cells reached 90%confluence, cultures were expanded to 175 cm² flasks until sufficientcells are available for injection. 10,000 cancer cells in PBS with 20%matrigel were subcutaneously into the right flank of each mouse The dayon which the tumor was implanted is designated as Day 0.

Treatment Regimen. Thirty (30) C57BL/6 mice (Charles River, female, 6-8wks old) were used in this study. The animals were assigned to 3 studygroups of 10 mice randomly, and housed as described herein. The antibodydosing regimen is shown in Table E4 below; in brief animals receivedintraperitoneal injections of the control IgG antibody, positive controlantibody (Bevacizumab), and anti-NRP2 antibody (as described herein),administered according to the protocol below (Table E4).

TABLE E4 GROUP TREATMENTS Route of Test Dose Admin. Group #MiceMaterials (mg/kg) (ROA) Frequency 1 10 Control IgG 25 IP BIW 2 10aNRP2-28 25 IP BIW 3 10 Bevacizumab 5 IP BIW * IP = Intraperitonealinjection

Summary of results. Animals bearing B16-F10 tumors and treated withthree doses of the mouse surrogate antibody aNRP2-28, which recognizesmouse NRP2 (and is a functional surrogate for anti-human NRP2 antibodyaNRP2-10v10). Treatment with aNRP2-28 resulted in tumor growthinhibition compare to the IgG control group, reaching statisticalsignificance (p<0.05) on Day 14, 16 and 19 (FIG. 19A). Accordingly,aNRP2-28 treated mice had the lowest terminal tumor weights (FIG. 19B).In addition, the anti-NRP2 antibody had more potent anti-tumor activityin this study than the VEGF-a blocking antibody Bevacizumab that waschosen as the positive control. There was no evidence of toxicity fromanimal body weight measurements and observations during the study (datanot shown). These results demonstrate that anti-NRP2 antibodies haveclear potential for therapeutic impact on cancer growth in this murinemelanoma model system.

Example 10 Evaluation of Antimetastatic Activity of Test CompoundaNRP2-28 in a Model of Spontaneous Metastasis

Studies were performed to investigate the in vivo anti-metastaticpotential of antibody aNRP2-10v10 using a surrogate antibody, aNRP2-28,which recognizes mouse NRP2 at a similar epitope region as antibody,aNRP2-10v10 (which does not cross react with mouse NRP2) in 4T1 cells (amelanoma cancer model) prepared as described below. The test antibodydosing regimen was initiated one day before cell injection and animalweights and tumor measurements were recorded three times a week untilstudy termination.

Mouse 4T1 cell line was purchased from ATCC. The cells were cultured in75 cm² flasks containing RPMI-1640 media supplemented with 10% FBS andincubated at 37° C. in humidified atmosphere of 5% CO₂. As cells reached90% confluence, cultures were expanded to 150 cm² flasks untilsufficient cells are available for injection. 50,000 cancer cells in PBSwere implanted subcutaneously into the right flank of each mouse.

Treatment Regimen. Twenty (20) Balb/c mice (Charles River, female, 5-6wks old) were used in this study. The animals were assigned to 3 studygroups of 10 mice randomly, and housed as described herein. The antibodydosing regimen is shown in Table E5 below; in brief animals receivedintraperitoneal injections of 10 mg/kg of the control IgG antibody, andaNRP2-28 (as described herein), administered according to the protocolbelow (Table E5); starting one day before cancer cell implantation untiltermination on day 23).

TABLE E5 GROUP TREATMENTS Route of Test Dose Admin. Group #MiceMaterials (mg/kg) (ROA) Frequency 1 10 Control IgG 10 IP days −1, 2, 6,9, 13, 15, 20, 23 2 10 aNRP2-28 10 IP days −1, 2, 6, 9, 13, 15, 20, 23 *IP = Intraperitoneal injection

Summary of results. Animals bearing 4T1 tumors and treated with mousesurrogate antibody aNRP2-28, which recognizes mouse NRP2 (and is afunctional surrogate for anti-human NRP2 antibody aNRP2-10v10)throughout the study showed a reduction in metastatic nodules in thelung at termination, reaching statistical significance (p≤0.05) (FIG.20A). Accordingly, there is a linear correlation between terminalantibody concentration and number of metastatic nodules (FIG. 20B).There was no evidence of toxicity from animal body weight measurementsand observations during the study (data not shown). These resultsdemonstrate that anti-NRP2 antibodies have clear potential forprophylactic, and potentially therapeutic impact on development ofdistant metastasis in this model system.

Example 11 Evaluation of Anti-Tumor Activity of Test Compounds inCombination with Cisplatin in a Xenograft Model of Triple-NegativeBreast Cancer

Studies were performed to investigate the in vivo anticancer potentialof the combination of the mouse surrogate antibody aNRP2-28, whichrecognizes mouse NRP2 (to block endogenous mouse NRP2) in combinationwith the anti-human NRP2 antibody aNRP2-10v10 (to block NRP2 on thehuman MDA-MB-231 cells) in combination with cisplatin in a humanxenograft mouse model using MDA-MB-231 cells (Triple Negative BreastCancer, TNBC cancer model). The dosing regimen was initiated when themean tumor volume of all study animals reached 50-100 mm³.

Treatment Regimen. Twenty (20) NSG mice (Jackson Laboratory, female, 5-6wks old) were used in this study. The animals were assigned to two studygroups of 10 mice randomly. The MDA-MB-231 cell line was prepared forinjection as described herein. The dosing regimen is shown in Table E6below; In brief animals received injections of 25 mg/kg of each themouse surrogate antibody aNRP2-28 and aNRP2-10v10 or IgG control, whichwere administered to mice intraperitoneally according to the protocolbelow (Table E6).

Human MDA-MB-231 cell line was purchased from ATCC. The cells werecultured in 75 cm2 flasks containing DMEM media supplemented with 10%fetal bovine serum (FBS) and incubated at 37° C. in humidifiedatmosphere of 5% CO2. As cells reached 90% confluence, cultures wereexpanded to 175 cm2 flasks until sufficient cells are available forinjection. 1.25×10⁶ cancer cells in PBS with 20% matrigel were injectedorthotopically into the mammary fat pad. The day on which the tumor wasimplanted is designated as Day 0.

TABLE E6 GROUP TREATMENTS Dose Group #Mice Materials (mg/kg) ROAFrequency 1 10 Control IgG/ 25/1-5 IP day 19 - Cisplatin termination 210 aNRP2-10/aNRP2-28/ 25/25/ IP day 19 - Cisplatin 1-5 termination * IP= Intraperitoneal injection

Summary of results. There was significant loss in body weight observedin both groups likely due to cisplatin-associated toxicity. Addition ofthe anti-NRP2 antibodies to the cisplatin treatment regimen increasedthe tumor-inhibitory effect of cisplatin starting on day 40 postinoculation, reaching statistical significance on day 60 (FIG. 21 ).These results demonstrate that targeting NRP2 in conjunction withcisplatin treatment enhances the chemotherapy efficacy in thisexperimental model of TNBC.

Example 12 Evaluation of Anti-Tumor Activity of aNRP2-10 in Combinationwith Flourouracil in a Xenograft Model of Non-Small Cell Lung Cancer

This study aimed to investigate the in vivo anticancer potential ofaNRP2-10v10 in combination with fluorouracil (5-FU) in a human xenograftmouse model using A549 cells (NSCLC cancer model). The dosing regimenwas initiated when the mean tumor volume of all study animals reached50-100 mm³ on day 16.

Treatment Regimen. Twenty (20) Nu/J mice (Jackson Laboratory, female,6-8 wks old) were used in this study. The animals were assigned to twostudy groups of 10 mice randomly. The A549 cell line was prepared forinjection as described herein. The dosing regimen is shown in Table E7below; In brief animals received injections of 12.5 mg/kg of aNRP2-10 orIgG control, which were administered to mice intraperitoneally twice aweek according to the protocol below (Table E7). Both groups alsoreceived 2.5 mg/kg of 5-FU three times a week.

Human A549 cell line was purchased from ATCC. The cells were thawed andcultured in 75 cm² flasks containing DMEM media supplemented with 10%FBS and incubated at 37° C. in humidified atmosphere of 5% CO₂. As cellsreached 90% confluence, cultures were expanded to 150 cm² flasks untilsufficient cells are available for injection. 2×10⁶ cancer cells in PBSwith 20% matrigel were subcutaneously implanted into right flank of eachmouse. The day on which the tumor was implanted is designated as Day 0.

TABLE E7 GROUP TREATMENTS Dose Group #Mice Materials (mg/kg) ROAFrequency 1 10 Control IgG/5-FU 12.5/2.5 IP BIW/TIW from day 16 on 2 10aNRP2-10/5-FU 12.5/2.5 IP BIW/TIW from day 16 on * IP = Intraperitonealinjection

Summary of results. The combination of 5-FU and aNRP2-10 performedbetter than 5-FU with control IgG, suggesting that targeting NRP2increases the efficacy of the chemotherapeutic drug 5-FU in this modelof NSCLC. A statistically significant difference was reached on day 51post cell inoculation (see FIG. 22 ). No obvious increased toxicity wasobserved in the aNRP2-10v10/5-FU group as determined by clinicalobservations and body weights.

Example 13 Evaluation of Anti-Lymphangiogenic Activity of aNRP2-10 in anExperimental Model of Neovascularization

To evaluate the anti-lymphangiogenic activity of the mouse surrogateantibody aNRP2-28, which recognizes mouse NRP2 (and is a functionalsurrogate for anti-human NRP2 antibody aNRP2-10v10) it was tested in anexperimental model of neovascularization.

Study Design & Treatment Regimen. The study consisted of 40 femaleC57BL/6 mice separated into four groups. Prior to study, animals wererandomized into four groups, 10 mice per group. On Day 0,neovascularization (NV) of the cornea was induced from alkali-burninjury in the eyes of animals under anesthesia. Animals were treatedintraperitoneally with control IgG (Group 1), aNRP2-28 (Group 2) andBevacizumab (Group 3) on post-injury Day 0, 3, 7 and 10. During thein-life phase, animals were monitored with daily clinical observationsand weekly body weights. Additionally, ocular assessments of injuredeyes were scored and photos taken on post-injury Day 7 and Day 14. Allanimals were euthanized on Day 14. Injured eyes from all animals wereremoved and dissected. Whole-mounted corneas were fixed, stained withLYVE-1, and analyzed immunohistochemically to examine the efficacy ofTest Articles.

Summary of results. The results in FIGS. 23A-23B demonstrate thataNRP2-28 can inhibit neolymphangiogenesis in an experimental model ofcorneal injury. FIG. 23A shows representative image scans of Lyve-1stained corneas of the IgG control and aNRP2-28 group. Plotting the meanvalues of % Lye-1* area for each treatment group. FIG. 23B shows a cleartrend towards reduced sprouting of lymphatic vessels in the aNRP2-10treatment groups as compared to the IgG control group. The observedeffect was close to statistical significance (p=0.07). These data showthat systemic administration of anti-NRP2 antibodies, such as aNRP2-28and its anti-human surrogate shows pharmacologic activity in anexperimental model of neovascularization.

All publications, patent applications, and issued patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or issued patent were specifically andindividually indicated to be incorporated by reference.

The invention claimed is:
 1. A therapeutic composition, comprising aphysiologically-acceptable carrier and at least one antibody orantigen-binding fragment thereof that specifically binds to a humanneuropilin-2 (NRP2) polypeptide (anti-NRP2 antibody), wherein the atleast one antibody or antigen-binding fragment thereof comprises: aheavy chain variable region (V_(H)) sequence that comprisescomplementary determining region V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3sequences; and a light chain variable region (V_(L)) sequence thatcomprises complementary determining region V_(L)CDR1, V_(L)CDR2, andV_(L)CDR3 sequences, wherein: the V_(H)CDR1, V_(H)CDR2, and V_(H)CDR3sequences comprise SEQ ID NOs: 19-21, respectively, and the V_(L)CDR1,V_(L)CDR2, and V_(L)CDR3 sequences comprise SEQ ID NOs: 22-24,respectively.
 2. The therapeutic composition of claim 1, wherein: theV_(H) sequence comprises a sequence at least 95% identical to SEQ ID NO:49, and the V_(L) sequence comprises a sequence at least 95% identicalto SEQ ID NO:
 50. 3. The therapeutic composition of claim 1, wherein theat least one antibody or antigen-binding fragment thereof comprises anIgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (includingsubclasses IgG1, IgG2, IgG3, and IgG4), IgM Fc domain, or variantthereof, optionally wherein the Fc domain is a human Fc domain.
 4. Thetherapeutic composition of claim 3, wherein the at least one antibody orantigen-binding fragment thereof comprises an IgG1 or IgG3 Fc domain. 5.The therapeutic composition of claim 3, wherein the at least oneantibody or antigen-binding fragment thereof comprises an IgG2 or IgG4Fc domain.
 6. The therapeutic composition of claim 5, wherein the atleast one antibody or antigen-binding fragment thereof comprises an IgG1or IgG4 Fc domain, optionally selected from SEQ ID NOs: 144-148.
 7. Thetherapeutic composition of claim 1, wherein the at least one antibody orantigen-binding fragment thereof comprises a modified IgG1 or IgG4 Fcdomain which has altered binding to FcRn, optionally wherein themodified IgG1 or IgG4 Fc domain comprises any one or more of YD(M252Y/T256D), DQ (T256D/T307Q), DW (T256D/T307W), YTE(M252Y/S254T/T256E), AAA (T307A/E380A/N434A), LS (M428L/N434S), M252Y,T256D/E, K288D/N, T307Q/W, E380C, N434F/Y, and/or Y436H/N/W mutations(EU numbering).
 8. The therapeutic composition of claim 1, wherein theat least one antibody or antigen-binding fragment thereof is amonoclonal antibody, optionally a humanized antibody.
 9. The therapeuticcomposition of claim 1, wherein the at least one antibody orantigen-binding fragment thereof is an Fv fragment, a single chain Fv(scFv) polypeptide, a minibody, or a unibody.
 10. The therapeuticcomposition of claim 1, wherein the composition has a purity of at leastabout 80% on a protein basis with respect to the at least one antibodyor antigen-binding fragment, and is substantially aggregate-free. 11.The therapeutic composition of claim 1, wherein the therapeuticcomposition is substantially endotoxin-free.
 12. The therapeuticcomposition of claim 1, wherein the therapeutic composition is asterile, injectable solution, optionally suitable for intravenous,intramuscular, subcutaneous, or intraperitoneal administration.
 13. Thetherapeutic composition of claim 1, which is a concentrated lyophilizedcomposition.